Modulating the alternative complement pathway

ABSTRACT

Provided herein are compositions, including pharmaceutical compositions, and methods for modulating, i.e., stimulating or inhibiting, activity of the alternative complement pathway, and methods of identifying factor H-binding proteins.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.14/571,102, filed Dec. 15, 2014, which is a continuation of Ser. No.13/120,125, filed Mar. 21, 2011, issued as U.S. Pat. No. 8,937,046,which in turn is a national stage of International Application No.PCT/US2009/057912, filed Sep. 22, 2009, which claims the benefit of U.S.Provisional Application No. 61/099,173, filed Sep. 22, 2008, the entirecontents of which are incorporated herein by reference in theirentireties and for all purposes.

STATEMENT OF RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH OR DEVELOPMENT

This invention was made with government support under grant numbers K08DK064790, R01 AI031005 and R01 DK076690 awarded by the NationalInstitutes of Health. The government has certain rights in theinvention.

SEQUENCE LISTING

The present disclosure contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Jun. 21, 2018, isnamed 53542-703-303.TXT and is 98, 671 bytes.

BACKGROUND OF THE INVENTION

The alternative pathway of complement is a phylogenetically ancient armof the innate immune system that eliminates invasive pathogens andfacilitates the removal of injured host cells. See J. M. Thurman et al.,J. Immunol. (2006) 176:1305-1310. The alternative pathway is continuallyauto-activated in the fluid phase, forming C3b which can bind to nearbybiologic surfaces. This spontaneously formed C3b then catalyzes furtheractivation and amplification through the alternative complement pathwayunless controlled by complement regulatory proteins (CRPs). The CRPsdissociate the alternative pathway C3 convertase (C3bBb) and/or serve ascofactors for the cleavage of C3 by factor I, forming iC3b. Thus,complement inhibition by CRPs on host cells is critical for protectinghost cells from spontaneous alternative complement pathway-mediatedinjury. Expression of CRPs is a fundamental mechanism by which thealternative pathway distinguishes healthy cells from injured cells andinvasive pathogens.

The endogenous membrane-bound proteins that control alternative pathwayactivation are decay-accelerating factor (DAF/CD55), membrane cofactorprotein (MCP/CD46), and complement receptor 1 (CR1). Other endogenousproteins that control alternative pathway activation include Factor H, acirculating ˜155 kD glycoprotein that regulates alternative pathwayactivation in the fluid phase as well as on tissue surfaces. See J. J.Alexander et al., Mol. Immunol. (2006) 44:123-132. Uncontrolledalternative pathway activation has been implicated in the pathogenesisof a diverse group of diseases, including age-related maculardegeneration (AMD), atypical hemolytic uremic syndrome (aHUS), type IImembranoproliferative glomerulonephritis (MPGN II), asthma, and renalischemia/reperfusion (I/R) injury. See J. M. Thurman et al., J. Immunol.(2006) 176:1305-1310. Injury to host tissues by the alternative pathwayindicates insufficient local control of the alternative pathway by thetarget tissue. Indeed, recent studies have demonstrated that mutationsin CRPs are strong risk factors for aHUS (M. C. Pickering et al., J.Exp. Med. (2007) 204:1249-1256) and MPGN II (R. J. Smith et al., J. Am.Soc. Nephrol. (2007) 18:2447-2456), and functional polymorphisms infactor H, a circulating regulator of the alternative pathway, areassociated with the development of AMD (R. J. Klein et al., Science(2005) 308:385-389; A. O. Edwards et al., Science (2005) 308:421-424; J.L. Haines et al., Science (2005) 308:419-421; G. S. Hageman et al.,Proc. Nat'l Acad. Sci. USA (2005) 102:7227-7232).

Ischemic acute kidney injury (AKI) in rodents (J. M. Thurman et al., J.Immunol. (2003) 170:1517-1523; J. M. Thurman et al., J. Am. Soc.Nephrol. (2006) 17:707-715) and in humans (J. M. Thurman et al., KidneyInt. (2005) 67:524-530) is associated with activation of the alternativepathway on the basolateral surface of injured tubular cells. We havefound that Complement receptor 1-related gene/protein y (Crry, a rodentanalog of human MCP and CR1) is the only CRP expressed by proximaltubular epithelial cells in mice, and that ischemia/reperfusion causesreduced surface expression of this protein. See J. M. Thurman et al., J.Clin. Invest. (2006) 116:357-368. Mice with congenital deficiency ofCrry (Crry+/−) are more sensitive than wild-type controls to ischemicacute renal failure (Id.), highlighting the importance of basolateralCrry for controlling the alternative pathway on this surface. It is notyet known whether polymorphisms or mutations in the CRPs may conferincreased risk of developing AKI in humans. Nevertheless, uncontrolledactivation of the alternative pathway in the setting of reduced surfaceCrry indicates that circulating factor H has a limited ability toprotect the surface of hypoxic tubular epithelial cells.

Factor H circulates in high concentrations (>400-600 μg/ml) and is apotent inhibitor of the alternative complement pathway. See J. J.Alexander et al., Mol. Immunol. (2006) 44:123-132. Alternative pathwayinhibition on cell surfaces by factor H, however, requires that itproperly bind to that surface. Several regions within the factor Hprotein bind to anionic surfaces, such as membranes rich in heparinsulfate or sialic acid, as well as to C3b on the surface. See S. Meri etal., Proc. Nat'l Acad. Sci. USA (1990) 87:3982-3986; M. K. Pangburn etal., J. Immunol. (2000) 164:4742-4751. Activation of the alternativepathway on a particular surface is strongly influenced by the affinityof factor H for that surface. The polymorphisms and mutations associatedwith AMD and aHUS, respectively, most frequently involve the region offactor H required for binding anionic surfaces and not the complementregulatory region. See M. C. Pickering et al., J. Exp. Med. (2007)204:1249-1256; A. P. Sjoberg et al., J. Biol. Chem. (2007)282:10894-10900. Thus, certain tissues or cell types require factor H toregulate alternative pathway activation on their surface. Differentbinding regions of the factor H protein may be necessary for complementregulation on those tissues or cell types. In some cases, the binding offactor H to surfaces in particular tissues may be affected by otherproteins. Identification of putative tissue-specific binding partners offactor H may provide potential mechanisms for modulating, i.e.,stimulating or inhibiting, activity of the alternative complementpathway in different tissues.

The disclosures of all publications, patents, patent applications andpublished patent applications referred to herein are hereby incorporatedherein by reference in their entirety and for all purposes.

BRIEF SUMMARY OF THE INVENTION

This application pertains to methods and compositions for modulating,e.g., stimulating or inhibiting, activity of the alternative complementpathway.

Provided herein are methods of modulating alternative complementactivity in an individual, comprising administering to the individual acomposition selected from the group consisting of (a) annexin A2 or abiologically-active fragment thereof; (b) a fusion protein comprising ananti-annexin A2 antibody or an antigen-binding fragment thereof fused toa complement inhibitor selected from the group consisting of DAF, factorH, MCP, CD59, CR1, and mouse Crry protein or a biologically-activefragment thereof; and (c) a biologically-active fragment of factor Hlacking the complement regulatory domain in SCRs 1 to 4 of full-lengthfactor H. In certain embodiments, the individual is a mammal. In certainembodiments, the mammal is a human, a mouse, or a rat. In certainembodiments, alternative complement activity is inhibited in anindividual. In certain embodiments, alternative complement activity isstimulated in an individual.

In certain embodiments, alternative complement activity is inhibited inan individual and the individual is administered a composition selectedfrom the group consisting of (a) annexin A2 or a biologically-activefragment thereof; and (b) a fusion protein comprising an anti-annexin A2antibody or antigen-binding fragment thereof fused to a complementinhibitor selected from the group consisting of DAF, factor H, MCP,CD59, CR1, and mouse Crry protein or a biologically-active fragmentthereof. In certain embodiments, the individual is administered acomposition comprising annexin A2 or a biologically-active fragmentthereof. In certain embodiments, the individual is administered acomposition comprising annexin A2. In certain embodiments, thecomposition is administered orally or by injection. In certainembodiments, the injection is intravenous.

In certain embodiments, alternative complement activity is stimulated inan individual and the individual is administered a composition anannexin A2 or a biologically-active fragment thereof. In certainembodiments, the individual is administered a composition comprisingannexin A2 or a biologically-active fragment thereof. In certainembodiments, the individual is administered a composition comprisingannexin A2. In certain embodiments, the composition is administeredorally or by injection. In certain embodiments, the injection isintravenous.

In certain embodiments, the individual is administered a compositioncomprising a fusion protein comprising an anti-annexin A2 antibody orantigen-binding fragment thereof fused to DAF or a biologically-activefragment thereof. In certain embodiments, the antigen-binding fragmentthereof comprises an Fab, Fab′, or F(ab′)₂ fragment. In certainembodiments, the biologically-active fragment of DAF fused to ananti-annexin A2 antibody or antigen-binding fragment thereof comprisesthe mature human DAF protein (amino acids 35-353 of SEQ ID NO: 17)without its GPI anchor. In certain embodiments, the biologically-activefragment of DAF fused to an anti-annexin A2 antibody or antigen-bindingfragment thereof comprises short consensus repeat sequences 1 to 4 (SCRs1 to 4) of full-length human DAF (amino acids 35 to 285 of SEQ IDNO:17). In certain embodiments, the composition is administered orallyor by injection. In certain embodiments, the injection is intravenous.

In certain embodiments, the individual is administered a compositioncomprising a fusion protein comprising an anti-annexin A2 antibody orantigen-binding fragment thereof fused to factor H or abiologically-active fragment thereof. In certain embodiments, theantigen-binding fragment thereof comprises an Fab, Fab′, or F(ab′)₂fragment. In certain embodiments, the biologically-active fragment offactor H fused to an anti-annexin A2 antibody or antigen-bindingfragment thereof comprises SCRs 1 to 4 of full-length factor H (aminoacids 21-266 of SEQ ID NO:3). In certain embodiments, thebiologically-active fragment of factor H fused to an anti-annexin A2antibody or antigen-binding fragment thereof comprises SCRs 1 to 8 offull-length factor H (amino acids 21-509 of SEQ ID NO:3). In certainembodiments, the biologically-active fragment of factor H fused to ananti-annexin A2 antibody or antigen-binding fragment thereof comprisesSCRs 1 to 18 of full-length factor H (amino acids 21-1106 of SEQ IDNO:3). In certain embodiments, the composition is administered orally orby injection. In certain embodiments, the injection is intravenous.

In certain embodiments, the individual is administered a compositioncomprising a fusion protein comprising an anti-annexin A2 antibody orantigen-binding fragment thereof fused to MCP or a biologically-activefragment thereof. In certain embodiments, the antigen-binding fragmentthereof comprises an Fab, Fab′, or F(ab′)₂ fragment. In certainembodiments, the biologically-active fragment of MCP fused to ananti-annexin A2 antibody or antigen-binding fragment thereof comprisesthe extracellular domain of human MCP (amino acids 35-343 of SEQ IDNO:19). In certain embodiments, the biologically-active fragment of MCPfused to an anti-annexin A2 antibody or antigen-binding fragment thereofcomprises SCRs 1 to 4 of full-length human MCP (amino acids 35-285 ofSEQ ID NO:19). In certain embodiments, the composition is administeredorally or by injection. In certain embodiments, the injection isintravenous.

In certain embodiments, the individual is administered a compositioncomprising a fusion protein comprising an anti-annexin A2 antibody orantigen-binding fragment thereof fused to CD59 or a biologically-activefragment thereof. In certain embodiments, the antigen-binding fragmentthereof comprises an Fab, Fab′, or F(ab′)₂ fragment. In certainembodiments, the biologically-active fragment of CD59 fused to ananti-annexin A2 antibody or antigen-binding fragment thereof comprisesthe extracellular domain of full-length human CD59 (amino acids 26-102of SEQ ID NO:21) lacking its GPI anchor and/or the amino acid to whichit is attached (i.e., Asn-102). In certain embodiments, the compositionis administered orally or by injection. In certain embodiments, theinjection is intravenous.

In certain embodiments, the individual is administered a compositioncomprising a fusion protein comprising an anti-annexin A2 antibody orantigen-binding fragment thereof fused to CR1 or a biologically-activefragment thereof. In certain embodiments, the antigen-binding fragmentthereof comprises an Fab, Fab′, or F(ab′)₂ fragment. In certainembodiments, the biologically-active fragment of CR1 fused to ananti-annexin A2 antibody or antigen-binding fragment thereof comprisesthe complete extracellular domain of full-length human CR1 (SCRs 1 to30)(amino acids 42-1971 of SEQ ID NO:23). In certain embodiments, thebiologically-active fragment of CR1 fused to an anti-annexin A2 antibodyor antigen-binding fragment thereof comprises SCRs 1 to 4 of full-lengthhuman CR1 (amino acids 42-295 of SEQ ID NO:23). In certain embodiments,the biologically-active fragment of CR1 fused to an anti-annexin A2antibody or antigen-binding fragment thereof comprises SCRs 1 to 11 offull-length human CR1 (amino acids 42-745 of SEQ ID NO:23). In certainembodiments, the biologically-active fragment of CR1 fused to ananti-annexin A2 antibody or antigen-binding fragment thereof comprisesSCRs 1 to 18 of full-length human CR1 (amino acids 42-1195 of SEQ IDNO:23). In certain embodiments, the composition is administered orallyor by injection. In certain embodiments, the injection is intravenous.

In certain embodiments, the individual is administered a compositioncomprising a fusion protein comprising an anti-annexin A2 antibody orantigen-binding fragment thereof fused to mouse Crry protein or abiologically-active fragment thereof. In certain embodiments, theantigen-binding fragment thereof comprises an Fab, Fab′, or F(ab′)₂fragment. In certain embodiments, the biologically-active fragment ofmouse Crry protein fused to an anti-annexin A2 antibody orantigen-binding fragment thereof comprises the complete extracellulardomain of full-length mouse Crry protein (amino acids 41-405 of SEQ IDNO:25). In certain embodiments, the biologically-active fragment ofmouse Crry protein fused to an anti-annexin A2 antibody orantigen-binding fragment thereof comprises SCRs 1 to 5 of full-lengthmouse Crry protein (amino acids 83-400 of SEQ ID NO:25). In certainembodiments, the composition is administered orally or by injection. Incertain embodiments, the injection is intravenous.

In certain embodiments, the alternative complement activity isassociated with renal inflammation or a drusen-related disease. Incertain embodiments, the renal inflammation is associated withischemia/reperfusion injury, ischemic acute kidney injury, thromboticthrombocytopenic purpura (TTP), hemolytic uremic syndrome (“HUS”), oratypical hemolytic uremic syndrome (“aHUS”). In certain embodiments, thedrusen-related disease is selected from the group consisting ofage-related macular degeneration, type II membranoproliferativeglomerulonephritis (“MPGN II”) and amyloidosis.

In certain embodiments, alternative complement activity is stimulated inan individual and the individual is administered a composition selectedfrom the group consisting of a biologically-active fragment of factor Hlacking the complement regulatory domain in SCRs 1 to 4 of full-lengthfactor H. In certain embodiments, the biologically active fragment offactor H comprises SCRs 19 and 20.

Provided herein are compositions for modulating alternative complementactivity in an individual. In certain embodiments, alternativecomplement activity is inhibited. In certain embodiments, alternativecomplement activity is stimulated.

In certain embodiments, the composition for modulating alternativecomplement activity comprises (a) annexin A2 or a biologically-activefragment thereof; (b) a fusion protein comprising an anti-annexin A2antibody or an antigen-binding fragment thereof fused to a complementinhibitor selected from the group consisting of DAF, factor H, MCP,CD59, CR1, and mouse Crry protein or a biologically-active fragmentthereof; or (c) a biologically-active fragment of factor H lacking thecomplement regulatory domain in SCRs 1 to 4 of full-length factor H; anda pharmaceutically acceptable carrier suitable for administration to anindividual. In certain embodiments, alternative complement activity isinhibited. In certain embodiments, alternative complement activity isstimulated.

In certain embodiments, alternative complement activity is inhibited andthe composition comprises annexin A2 or a biologically-active fragmentthereof and a pharmaceutically acceptable carrier suitable foradministration to an individual. In certain embodiments, alternativecomplement activity is inhibited and the composition comprises annexinA2 and a pharmaceutically acceptable carrier suitable for administrationto an individual. In certain embodiments, the composition comprises ananti-annexin A2 antibody or an antigen-binding fragment thereof fused toa complement inhibitor selected from the group consisting of DAF, factorH, MCP, CD59, CR1, and mouse Crry protein or a biologically-activefragment thereof and a pharmaceutically acceptable carrier suitable foradministration to an individual. In certain embodiments, theantigen-binding fragment of an anti-annexin A2 antibody comprises anFab, Fab′, or F(ab′)₂ fragment.

In certain embodiments, alternative complement activity is stimulatedand the composition comprises annexin A2 or a biologically-activefragment thereof and a pharmaceutically acceptable carrier suitable foradministration to an individual. In certain embodiments, alternativecomplement activity is simulated and the composition comprises annexinA2 and a pharmaceutically acceptable carrier suitable for administrationto an individual.

In certain embodiments, the biologically-active fragment of DAFcomprises the mature human DAF protein (amino acids 35-353 of SEQ IDNO:17) without its GPI anchor or short consensus repeat sequences 1 to 4(SCRs 1 to 4) of full-length human DAF (amino acids 35 to 285 of SEQ IDNO:17). In certain embodiments, the biologically-active fragment offactor H comprises SCRs 1 to 4 of full-length factor H (amino acids21-266 of SEQ ID NO:3), SCRs 1 to 8 of full-length factor H (amino acids21-509 of SEQ ID NO:3), or SCRs 1 to 18 of full-length factor H (aminoacids 21-1106 of SEQ ID NO:3). In certain embodiments, thebiologically-active fragment of MCP comprises the extracellular domainof human MCP (amino acids 35-343 of SEQ ID NO:19) or SCRs 1 to 4 offull-length human MCP (amino acids 35-285 of SEQ ID NO:19). In certainembodiments, the biologically-active fragment of CD59 comprises theextracellular domain of full-length human CD59 (amino acids 26-102 ofSEQ ID NO:21) lacking its GPI anchor and/or the amino acid to which itis attached (i.e., Asn-102). In certain embodiments, thebiologically-active fragment of CR1 comprises the complete extracellulardomain of full-length human CR1 (SCRs 1 to 30)(amino acids 42-1971 ofSEQ ID NO:23), SCRs 1 to 4 of full-length human CR1 (amino acids 42-295of SEQ ID NO:23), SCRs 1 to 11 of full-length human CR1 (amino acids42-745 of SEQ ID NO:23), or SCRs 1 to 18 of full-length human CR1 (aminoacids 42-1195 of SEQ ID NO:23). In certain embodiments, thebiologically-active fragment of mouse Crry protein comprises thecomplete extracellular domain of full-length mouse Crry protein (aminoacids 41-405 of SEQ ID NO:25) or SCRs 1 to 5 of full-length mouse Crryprotein (amino acids 83-400 of SEQ ID NO:25).

In certain embodiments, alternative complement activity is stimulatedand the composition comprises a biologically-active fragment of factor Hlacking the complement regulatory domain in SCRs 1 to 4 of full-lengthfactor H and a pharmaceutically acceptable carrier suitable foradministration to an individual. In certain embodiments, thebiologically-active fragment of factor H comprises SCRs 19 and 20 (aminoacids 1109-1232 of SEQ ID NO:3).

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1E. Blockade of surface inhibition by factor H amplifies renalinjury after I/R. Mice were subjected to renal I/R. After four hours ofreperfusion the mice received a tail-vein injection of vehicle orrH19-20. (FIG. 1A) After eight hours of reperfusion the plasma C3alevels in mice treated with rH19-20 were higher than those that receivedvehicle. (FIG. 1B) After twenty-four hours of reperfusion the plasma C3alevels in mice treated with rH19-20 were higher than those that receivedvehicle, although the difference was no longer significant. FIG. 1C)Morphologic injury in mice that received vehicle demonstrated tubularinjury in the outer medulla, but the cortex (inset) was primarilyspared. (FIG. 1D) Mice that received rH19-20 developed severe tubularinjury in the outer medulla and severe injury extended into the cortex(inset). (FIG. 1E) After twenty-four hours of reperfusion, serum ureanitrogen (SUN) levels were also significantly higher in mice treatedwith rH19-20 than those that received vehicle.

FIGS. 2A-2B. Factor H levels increase in the kidney after I/R. Mice weresubjected to renal I/R and Western blot analysis was performed tomeasure levels of factor H within the kidney. (FIG. 2A) Levels of factorH within the kidney increased during reperfusion. (FIG. 2B) Levels offactor H were increased in both wild-type and factor B deficient (fB−/−)mice, demonstrating that tissue bound C3 fragments are not required forfactor H to bind within the tissue.

FIGS. 3A-3F. Tissue bound factor H does not co-localize with C3deposits. Mice were subjected to renal I/R, and immunofluorescencemicroscopy was performed to identify factor H and C3. (FIG. 3A) Atbaseline, isolated deposits of C3 were observed in thetubulointerstitium and little factor H was observed. After 8 hours (FIG.3B) and 24 hours (FIG. 3C) of reperfusion, increasing quantities of bothC3 and factor H were detected in the tubulointerstitium. (FIG. 3D) Ahigh-powered view of the kidney after 24 hours of reperfusiondemonstrates that factor H is localized over damaged tubules, but doesnot co-localize with tissue deposits of C3. (FIG. 3E) By 48 hours ofreperfusion, tissue bound factor H and C3 are similar to baselinelevels. (FIG. 3F) A section of a kidney at 24 hours of reperfusion wasstained with a secondary antibody only to demonstrate specificity of thestaining for C3 and factor H. Original magnification ×200 A-C, E, F and×400 D.

FIGS. 4A-3C. Purified factor H binds to Annexin A2 expressed in thekidney after I/R. Factor H was purified by heparin chromatography, andits purity was verified by Coomassie staining and Western blot analysis.(FIG. 4A) Protein lysates of un-manipulated kidneys and kidneyssubjected to I/R were separated by SDS-PAGE and transferred tonitrocellulose membrane. The membrane was incubated with biotinylatedfactor H to determine whether protein binding partners were present.Some binding partners appeared more abundant in the post-ischemiclysates (arrow). (FIG. 4B) Biotinylated factor H was next incubated withtissue lysates. Streptavidin beads were used to pull down the factor Halong with binding partners, and the proteins were examined by Coomassiestaining. Again, some binding partners appeared more abundant in lysatesfrom post-ischemic tissue (arrow). The ˜39 kD protein was digested withtrypsin and analyzed by reverse phase nanospray LC-MS/MS and wasidentified with high confidence as Annexin A2. (FIG. 4C) Typically, atleast 2 peptide hits are sought to confirm a protein's identity. Weobtained 6 unique peptides for annexin A2, and three were manuallyconfirmed. FIG. 4C shows the MS spectrum for one of the peptides:SLYYYIQQDTK (SEQ ID NO:31).

FIGS. 5A-5B. Annexin A2 is expressed on injured tubules after I/R, andfactor H pulls down annexin A2 in kidney lysates. Immunohistochemistryfor annexin A2 was performed on sections of kidneys subjected to I/R.(FIG. 5A) Annexin A2 was detected in glomeruli and around injuredtubules of the outer medulla. Original magnification ×400. (FIG. 5B)Biotinylated factor H was used to pull-down binding partners which werethen separated by SDS-PAGE and probed with a monoclonal antibody toannexin A2. Factor H bound to a greater abundance of annexin A2 in thelysates of post-ischemic kidneys.

FIG. 6. Surface plasmon resonance demonstrates that annexin A2 binds tofactor H with high affinity. Factor H was immobilized on a CM5 sensorchip. Bovine annexin A2 was diluted in Hank's Buffered Saline with orwithout 3 mM CaCl₂ and injected at the following concentrations: HBSalone (no protein)+3 mM CaCl₂ (second line from bottom-no binding); 3.7μg/ml in HBS+3 mM CaCl₂ (third line from bottom); 7.5 μg/ml in HBS+3 mMCaCl₂ (fourth line from bottom); 15 μg/ml in HBS+3 mM CaCl₂ (fifth linefrom bottom); 30 μg/ml in HBS+3 mM CaCl₂ (top line); and 30 μg/ml inHank's Buffered Saline without calcium (bottom essentially constantline-no binding). The analyte bound to the chip with high affinity (17nm), but rapidly dissociated from the chip when calcium was removed fromcell.

FIGS. 7A-7D. CR2-fH prevents complement activation after renal I/R.CR2-fH is a recombinant fusion protein that uses the C3d-binding regionof CR2 to target the complement-inhibitory region of factor H to sitesof C3d deposition. Mice were subjected to renal I/R and were theninjected with vehicle or with 250 μg of CR2-fH. (A-B) Immunofluorescencemicroscopy for C3 revealed that mice treated with vehicle (FIG. 7A)demonstrated tubular deposition of C3, whereas only sparse areas of C3were seen in mice treated with CR2-fH (FIG. 7B). Treatment with CR2-fHdecreased tubular deposition of C3 compared to control mice treated withvehicle alone (FIG. 7C) and attenuated the development of renal injuryafter I/R as measured by SUN (serum urea nitrogen) levels (FIG. 7D).

FIG. 8. Mice were treated with ASOs to Annexin A2 (Column “ASO (8hours)” or with control ASOs (Column “Control (8 hours)”), and were thensubjected to renal I/R. After eight hours of reperfusion plasma sampleswere obtained, and C3a was measured by ELISA, as described herein andknown in the art. Levels of C3a in mice treated with ASOs to Annexin A2were higher than those in control animals, demonstrating that Annexin A2functions to limit complement activation after renal I/R.

FIG. 9. Mice were treated with ASO to Annexin A2 or with control ASOs,and were then subjected to renal I/R. After twenty-four hours ofreperfusion the mice were sacrificed, and serum urea nitrogen (SUN) wasmeasured, by methods known in the art, as a marker of renal function.SUN levels in mice treated with ASOs to Annexin A2 were higher thanthose in control animals, demonstrating that Annexin A2 functions tolimit renal injury after renal I/R. See Column “ASO.:” SUN levels incomplement deficient mice (deficient in factor B, or fB^(−/−) mice) thatwere treated with the ASOs to Annexin A2 were not higher than controlmice, indicating that the ASOs to Annexin A2 require an intactcomplement system in order to cause renal injury after I/R. See Column“fB^(−/−) ASO.”

FIG. 10. Renal tubular epithelial cells were grown in culture and werethen exposed to 10% mouse serum (shaded curve). Treatment of the cellswith rH19-20 blocked protection of the cells by factor H present withinthe serum, thereby enhancing the deposition of C3 on the cell surface(black line). The addition of purified Annexin A2 to the cellsupernatant had a similar effect (dashed line). The addition of bothrH19-20 and Annexin A2 caused a similar degree of complement activationon the cells than was obtained with either reagent alone.

DETAILED DESCRIPTION OF THE SEQUENCES

SEQ ID NO:1 is the amino acid sequence of full-length human annexin A2protein.

SEQ ID NO:2 is the nucleotide sequence of full-length human annexin A2protein.

SEQ ID NO:3 is the amino acid sequence of full-length human factor H.

SEQ ID NO:4 is the nucleotide sequence of a cDNA encoding full-lengthhuman factor H.

SEQ ID NO:5 is the amino acid sequence of a fragment of human factor Hcomprising short consensus repeat sequences 1 to 4.

SEQ ID NO:6 is the amino acid sequence of a fragment of human factor Hcomprising short consensus repeat sequences 1 to 8.

SEQ ID NO:7 is the amino acid sequence of a fragment of human factor Hcomprising short consensus repeat sequences 1 to 18.

SEQ ID NO:8 is the amino acid sequence of a fragment of human factor Hcomprising short consensus repeat sequences 19 to 20.

SEQ ID NO:9 is the amino acid sequence of full-length mouse factor H.

SEQ ID NO:10 is the nucleotide sequence of a cDNA encoding full-lengthmouse factor H.

SEQ ID NO: 11 is the amino acid sequence of the signal peptide of humanCD5 protein.

SEQ ID NO: 12 is the nucleotide sequence of the signal peptide of humanCD5 protein.

SEQ ID NO:13 is the amino acid sequence of the signal peptide of humanCR2 protein, short version.

SEQ ID NO: 14 is the nucleotide sequence of the signal peptide of humanCR2 protein, short version.

SEQ ID NO:15 is the amino acid sequence of the signal peptide of humanCR2 protein, long version.

SEQ ID NO: 16 is the nucleotide sequence of the signal peptide of humanCR2 protein, long version.

SEQ ID NO: 17 is the amino acid sequence of human decay acceleratingfactor (DAF).

SEQ ID NO:18 is the nucleotide sequence of a cDNA encoding human decayaccelerating factor (DAF).

SEQ ID NO:19 is the amino acid sequence of human membrane cofactorprotein (MCP) protein.

SEQ ID NO:20 is the nucleotide sequence of a cDNA encoding humanmembrane cofactor protein (MCP) protein.

SEQ ID NO:21 is the amino acid sequence of human CD59 protein.

SEQ ID NO:22 is the nucleotide sequence of a cDNA encoding full-lengthhuman CD59 protein.

SEQ ID NO:23 is the amino acid sequence of human CR1 protein.

SEQ ID NO:24 is the nucleotide sequence of a cDNA encoding full-lengthhuman CR1 protein.

SEQ ID NO:25 is the amino acid sequence of mouse Crry protein.

SEQ ID NO:26 is the nucleotide sequence of a cDNA encoding full-lengthmouse Crry protein.

SEQ ID NO:27 to SEQ ID NO:29 are antisense oligodeoxynucleotides againstannexin 2 as described herein.

DETAILED DESCRIPTION OF THE INVENTION

The alternative pathway of complement is an important part of the innateimmune system, but uncontrolled alternative pathway activationcontributes to tissue injury in a wide variety of diseases includingrenal ischemia/reperfusion (I/R) and ischemic acute kidney injury.Factor H, a circulating alternative pathway regulator, cannot entirelyprevent alternative complement-mediated injury in the kidney after I/R,suggesting that factor H has limited efficacy at preventing complementactivation on the renal tubules, despite the fact that the tissue-boundlevel of native factor H increases during reperfusion. We demonstrateherein that factor H binds to annexin A2 in post-ischemic kidneys, andthat mice that do not express annexin A2 develop more severe injuryafter renal I/R. We conclude that the inability of native factor H toprevent complement activation on the renal tubules after I/R resultsfrom its insufficient binding affinity for renal epithelial cells, andthat expression of protein ligands for factor H during reperfusion iscritical to limiting alternative complement-mediated renal injuryresulting from I/R.

Definitions

General reference to “the composition” or “compositions” includes and isapplicable to compositions of the invention.

As used herein, the singular form of the articles “a,” “an,” and “the”includes plural references unless indicated otherwise. For example, thephrase “a biologically-active CR2 fragment” includes one or morebiologically-active CR2 fragments.

Reference to “about” a value or parameter herein includes (anddescribes) embodiments that are directed to that value or parameter perse. For example, description referring to “about X” includes descriptionof “X.”

It is understood that aspects and embodiments of the invention describedherein include consisting and/or consisting essentially of aspects andembodiments.

As used herein, the term individual refers to a vertebrate, preferably amammal, more preferably a human. Mammals include, but are not limitedto, farm animals, sport animals, pets, primates, mice and rats. Incertain embodiments, the individual is human. In certain embodiments,the individual is an individual other than a human. In certainembodiments, the individual is an animal model for the study of adisease in which the alternative complement pathway is implicated.

Provided herein are compositions and methods for modulating, i.e.,stimulating or inhibiting, activity of the alternative complementpathway, and methods of identifying factor H binding proteins.

Compositions for Modulating Activity of the Alternative ComplementPathway

In one aspect, there are provided a composition or compositions suitablefor modulating, i.e., stimulating or inhibiting, activity of thealternative complement pathway. In certain embodiments, the compositionsare suitable for inhibiting activity of the alternative complementpathway in the kidneys. In certain embodiments, the compositionssuitable for inhibiting activity of the alternative complement pathwaycomprise human annexin A2 protein (SEQ ID NOs:1 and 2) orbiologically-active fragments thereof.

The annexins are a family of calcium- (Ca²⁺—) and phospholipid-bindingproteins that differ from most other Ca²⁺-binding proteins in theirCa²⁺-binding sites. The annexin family Ca²⁺-binding site has a uniquearchitecture that enables annexin family members to reversibly dock ontothe periphery of cellular and/or organellar membranes. The conservedCa²⁺-binding site characteristic of annexin family members is located inthe annexin core domain, and comprises four annexin repeats, eachseventy (70) amino acids long. The annexin core domain is α-helical andforms a compact, curved disc with a convex surface comprising the Ca²⁺—and membrane-binding sites and a concave side oriented away from themembrane that is available for other types of interaction. Annexinfamily members also typically have an amino-terminal domain of variablelength that precedes the annexin core domain and is diverse in sequenceand structure. This variable domain mediates regulatory interactionswith protein ligands as well as the annexin-membrane association. See U.Rescher et al., J. Cell Sci. (2004) 117:2631-2639. Twelve annexinsubfamilies have been characterized in vertebrates, each havingdifferent splice variants, with different amino-terminal domains anddifferently positioned Ca²⁺-binding sites.

As used herein, the terms “annexin A2,” “annexin II,” or “annexin 2”refer to proteins of the annexin A2 subfamily, which have been shown toassociate with diverse sites of actin attachment at cell membranes, andto serve a receptors for plasminogen and tissue plasminogen activator,positively modulating the fibrinolytic cascade, among other activities.Id. Annexin A2 has also been identified as a component of drusen inmonkeys affected with both early- and late-onset macular degeneration.See S. Umeda et al., FASEB J. (2005) 19(12):1683-1685. Annexin A2 hasnot previously been shown to interact with any components of thealternative complement pathway, however. As used herein, the term“biologically-active fragment” of annexin A2, annexin II, or annexin 2,refers to a fragment of annexin A2 capable of interacting with orbinding to renal tubules in the kidney and interacting with or bindingto factor H or a biologically active fragment thereof. The ability of abiologically-active fragment of annexin A2 to interact with or bindrenal tubules or factor H can be assayed by a variety of routine methodsknown to those skilled in the art, including gel mobility shift assays,Western blot, immunoprecipitation, surface plasmon resonance, and thelike.

In certain embodiments, the compositions suitable for inhibitingactivity of the alternative complement pathway in the kidneys comprisehomologues of human annexin A2 protein (SEQ ID NOs: 1 and 2) orbiologically-active fragments thereof. A homologue of an annexin A2protein or biologically-active fragment thereof includes proteins whichdiffer from a naturally occurring human annexin A2 (or from abiologically-active fragment thereof) in that at least one or a few, butnot limited to one or a few, amino acids have been deleted (e.g., atruncated version of the protein, such as a peptide or fragment),inserted, inverted, substituted and/or derivatized (e.g., byglycosylation, phosphorylation, acetylation, myristoylation,prenylation, palmitation, amidation and/or addition ofglycosylphosphatidyl inositol). For example, a human annexin A2homologue may have an amino acid sequence that is at least about 70%identical to the amino acid sequence of a naturally occurring humanannexin A2 (e.g., SEQ ID NO:1), for example at least about any of 75%,76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to theamino acid sequence of a naturally occurring human annexin A2 (e.g., SEQID NO:1). In certain embodiments, a homologue of human annexin A2 (or abiologically-active fragment thereof) retains all the biologicalactivities of human annexin A2 (or a biologically-active fragmentthereof), i.e., the ability to bind renal tubules and to bind factor H.In certain embodiments, the homologue of human annexin A2 (or abiologically-active fragment thereof) retains at least about 50%, forexample, at least about any of 60%, 70%, 80%, 90%, or 95% of thebiological activity of annexin A2 (or a biologically-active fragmentthereof).

Amino acid sequence identity can be determined in various ways, forexample, using publicly available computer software such as BLAST,BLAST-2, ALIGN or MEGALIGN™ (DNASTAR) software. One skilled in the artcan determine appropriate parameters for measuring alignment, includingany algorithms needed to achieve maximal alignment over the full lengthof the sequences being compared.

In another aspect, the compositions suitable for inhibiting activity ofthe alternative complement pathway comprise a fusion protein comprisingan anti-annexin A2 antibody or antigen-binding fragment thereof fused toa complement inhibitor. In certain embodiments, the complement inhibitoris selected from the group consisting of decay accelerating factor(DAF), factor H, membrane cofactor protein (MCP), CD59, complementreceptor 1 (CR1), and mouse complement receptor 1-related gene/protein y(“Crry”).

As used herein, the term “decay accelerating factor,” “DAF,” or “CD55”refers to a seventy kilodalton (“kD”) membrane glycoprotein comprisingfour short consensus repeat (SCR) domains followed by a heavilyO-glycosylated serine/threonine-rich domain at the C-terminus thatelevates the molecule from the membrane surface, including homologuesthereof. DAF is anchored into the cell membrane by aglycosylphosphatidylinositol (“GPI”) anchor. DAF protects the cellsurface from complement activation by dissociating membrane-bound C3convertases that are required to cleave complement protein C3 and toamplify the alternative complement cascade.

SEQ ID NO:17 represents the full-length human DAF amino acid sequence(see, e.g., UniProtKB/Swiss-Prot. Accession No. P08173). Amino acids1-34 correspond to the signal peptide, amino acids 35-353 appear in themature protein, and amino acids 354-381 are removed from the polypeptideafter translation. Within the mature protein, amino acids 35-96correspond to SCR 1, amino acids 96-160 correspond to SCR 2, amino acids161-222 correspond to SCR 3, amino acids 223-285 correspond to SCR 4,and amino acids 287-353 correspond to the O-glycosylatedserine/threonine-rich domain. The GPI anchor is attached to DAF at aserine at position 353. It is understood that species and strainvariations exist for the disclosed peptides, polypeptides, and proteins,and that DAF or biologically-active fragments thereof encompasses allspecies and strain variations. As used herein, the term“biologically-active” fragment of DAF refers to any fragment of DAFlacking a GPI anchor and/or the amino acid to which it is attached(i.e., Ser-353), including any fragments of the full-length DAF proteincomprising, consisting essentially of or consisting of 1, 2, 3, or 4 SCRdomains, with or without the O-glycosylated serine/threonine-richdomain, having some or all the complement inhibitory activity of thefull-length DAF protein.

In certain embodiments, the compositions suitable for inhibitingactivity of the alternative complement pathway in the kidneys comprise afusion protein comprising an anti-annexin A2 antibody or antigen-bindingfragment thereof fused to full-length human decay-accelerating factor(DAF) (SEQ ID NOs:17 and 18) or a biologically-active fragment thereof.In certain embodiments, the biologically-active fragment of human DAFcomprises the mature human DAF protein (amino acids 35-353 of SEQ IDNO:17) without its GPI anchor or short consensus repeat sequences 1 to 4(SCRs 1 to 4) of full-length human DAF (amino acids 35 to 285 of SEQ IDNO:17). The anti-annexin A2 antibody or antigen-binding portion of thefusion protein is responsible for delivering the composition to renalsites of alternative complement activation by selectively binding toannexin A2 expressed in the kidneys, while the DAF portion of the fusionprotein is responsible for inhibiting activity of the alternativecomplement pathway.

In certain embodiments, the fusion protein comprises an anti-annexin A2antibody or antigen-binding fragment thereof fused to full-length humanDAF. In certain embodiments, the fusion protein comprises ananti-annexin A2 antibody or antigen-binding fragment thereof fused to abiologically active fragment of full-length human DAF. In certainembodiments, the fusion protein comprises an anti-annexin A2 antibody orantigen-binding fragment thereof fused to a biologically active fragmentof full-length human DAF comprising the full-length protein lacking itsGPI anchor (amino acids 35-353 of SEQ ID NO: 17). In certainembodiments, the fusion protein comprises an anti-annexin A2 antibody orantigen-binding fragment thereof fused to a biologically active fragmentof full-length human DAF comprising short consensus repeat sequences 1to 4 (SCRs 1 to 4)(amino acids 35 to 285 of SEQ ID NO:17).

A homologue of a human DAF protein or a biologically-active fragmentthereof includes proteins which differ from a naturally occurring humanDAF (or biologically-active fragment thereof) in that at least one or afew, but not limited to one or a few, amino acids have been deleted(e.g., a truncated version of the protein, such as a peptide orfragment), inserted, inverted, substituted and/or derivatized (e.g., byglycosylation, phosphorylation, acetylation, myristoylation,prenylation, palmitation, amidation and/or addition ofglycosylphosphatidyl inositol). For example, a human DAF homologue mayhave an amino acid sequence that is at least about 70% identical to theamino acid sequence of a naturally occurring human DAF (e.g., SEQ IDNO:17), for example at least about any of 75%, 76%, 77%, 78%, 79%, 80%,81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of anaturally occurring human DAF (e.g., SEQ ID NO:17). In certainembodiments, a homologue of human DAF (or a biologically-active fragmentthereof) retains all the alternative complement pathway inhibitoryactivity of human DAF (or a biologically-active fragment thereof). Incertain embodiments, the homologue of human DAF (or abiologically-active fragment thereof) retains at least about 50%, forexample, at least about any of 60%, 70%, 80%, 90%, or 95% of thecomplement inhibition activity of human DAF (or a biologically-activefragment thereof).

As used herein, the term “complement factor H,” “factor H,” or “FH”refers to complement factor H, a single polypeptide chain plasmaglycoprotein, including homologues thereof. The protein is composed of20 conserved short consensus repeat (SCR) domains of approximately 60amino acids, arranged in a continuous fashion like a string of beads,separated by short linker sequences of 2 to 6 amino acids each. Factor Hbinds to C3b, accelerates the decay of the alternative pathwayC3-convertase (C3bBb), and acts as a cofactor for the proteolyticinactivation of C3b. In the presence of factor H, C3b proteolysisresults in the cleavage of C3b. Factor H has at least three distinctbinding domains for C3b, which are located within SCRs 1-4, SCRs 5-8,and SCRs 19-20. Each site of factor H binds to a distinct region withinthe C3b protein: the N-terminal sites bind to native C3b; the secondsite, located in the middle region of factor H, binds to the C3cfragment and the site located within SCR19 and 20 binds to the C3dregion. In addition, factor H also contains binding sites for heparin,which are located within SCR 7, SCRs 5-12, and SCR 20 of factor H andoverlap with those of the C3b binding sites. Structural and functionalanalyses have shown that the domains for the complement inhibitoryactivity of factor H are located within the first four N-terminal SCRdomains.

SEQ ID NO:3 represents the full-length human factor H amino acidsequence (see, e.g., UniProtKB/Swiss-Prot. Accession No. P08603). Aminoacids 1-18 correspond to the signal peptide, and amino acids 19-1231correspond to the mature protein. Within that protein, amino acids 21-80correspond to SCR 1, amino acids 85-141 correspond to SCR 2, amino acids146-205 correspond to SCR 3, amino acids 210-262 correspond to SCR 4,and amino acids 267-320 correspond to SCR 5. The full-length mousefactor H amino acid sequence is represented herein by SEQ ID NO:9 (see,e.g., UniProtKB/Swiss-Prot. Accession No. P06909). Amino acids 1-18correspond to the signal peptide, and amino acids 19-1234 correspond tothe mature protein. Within that protein, SCRs 1 and 2 domains of mousefactor H protein are located within the mouse factor H amino sequence atpositions 19-82 of SEQ ID NO:9 (SCR 1) and positions 83-143 of SEQ IDNO:9 (SCR 2). Human and mouse factor H are approximately 61% identicalover the full length amino acid sequences represented by SEQ ID NO:3 andSEQ ID NO:9. It is understood that species and strain variations existfor the disclosed peptides, polypeptides, and proteins, and that factorH or biologically-active fragments thereof encompasses all species andstrain variations.

As used herein, the term “biologically-active” fragment of factor Hrefers to any portion of a factor H protein having some or all thecomplement inhibitory activity of the full-length factor H protein, andincludes, but is not limited to, factor H fragments comprising SCRs 1 to4, SCRs 1 to 8, SCRs 1 to 18, SCRs 19 to 20, or any homologue of anaturally-occurring factor H or fragment thereof, as described in detailbelow. In certain embodiments, the biologically-active fragment offactor H has one or more of the following properties: (1) binding toC-reactive protein (CRP), (2) binding to C3b, (3) binding to heparin,(4) binding to sialic acid, (5) binding to endothelial cell surfaces,(6) binding to cellular integrin receptor, (7) binding to pathogens, (8)C3b co-factor activity, (9) C3b decay-acceleration activity, and (10)inhibiting the alternative complement pathway.

In certain embodiments, the compositions suitable for inhibitingactivity of the alternative complement pathway in the kidneys comprise afusion protein comprising an anti-annexin A2 antibody or antigen-bindingfragment thereof fused to human factor H (SEQ ID NOs:3 and 4) or abiologically-active fragment thereof. In certain embodiments, thebiologically-active fragment of human factor H comprises short consensusrepeat sequences 1 to 4 (SCRs 1 to 4)(SEQ ID NO:5), short consensusrepeat sequences 1 to 8 (SCRs 1 to 8)(SEQ ID NO:6), or short consensusrepeat sequences 1 to 18 (SCRs 1 to 18)(SEQ ID NO:7). The anti-annexinA2 antibody or antigen-binding portion of the fusion protein isresponsible for delivering the composition to renal sites of alternativecomplement activation by selectively binding to annexin A2 expressed inthe kidneys, while the factor H portion of the fusion protein isresponsible for inhibiting activity of the alternative complementpathway.

In certain embodiments, the fusion protein comprises an anti-annexin A2antibody or antigen-binding fragment thereof fused to full-length humanfactor H. In certain embodiments, the fusion protein comprises ananti-annexin A2 antibody or antigen-binding fragment thereof fused to abiologically active fragment of full-length human factor H comprisingSCRs 1 to 4. In certain embodiments, the fusion protein comprises ananti-annexin A2 antibody or antigen-binding fragment thereof fused to abiologically active fragment of full-length human factor H comprisingSCRs 1 to 8. In certain embodiments, the fusion protein comprises ananti-annexin A2 antibody or antigen-binding fragment thereof fused to abiologically active fragment of full-length human factor H comprisingSCRs 1 to 18.

In certain embodiments, the biologically-active fragment of factor Hcomprises the first four N-terminal SCR domains of factor H. In certainembodiments, the biologically-active fragment of factor H comprises thefirst five N-terminal SCR domains of factor H. In certain embodiments,the biologically-active fragment of factor H comprises the first sixN-terminal SCR domains of factor H. In certain embodiments, thebiologically-active fragment of factor H comprises the first eightN-terminal SCR domains of factor H. In certain embodiments, thebiologically-active fragment of factor H comprises the first eighteenN-terminal SCR domains of factor H. In certain embodiments, thebiologically-active fragment of factor H comprises SCRs 1 to 4 of factorH. In certain embodiments, the biologically-active fragment of factor Hcomprises SCRs 1 to 8 of factor H. In certain embodiments, thebiologically-active fragment of factor H comprises SCRs 1 to 18 offactor H. In certain embodiments, the biologically-active factor Hfragment comprises (and in certain embodiments consists of or consistsessentially of) at least the first four N-terminal SCR domains of factorH, including for example, at least any of the first 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, or more N-terminal SCR domains of factorH.

In certain embodiments, the biologically-active fragment of factor H isderived from a wild-type factor H. In certain embodiments, thebiologically-active fragment of factor H is derived from anaturally-occurring protective variant of factor H.

In certain embodiments, the biologically-active fragment of factor Hlacks a heparin binding site. This can be achieved, for example, bymutation of the heparin binding site on a biologically-active fragmentof factor H, or by selecting biologically-active factor H fragments thatdo not contain a heparin binding site. In certain embodiments, thebiologically-active fragment of factor H has a polymorphism that isprotective to age-related macular degeneration. See Hageman et al.,Proc. Nat'l Acad. Sci. USA 102(20):7227.

A homologue of a human factor H protein or a biologically-activefragment thereof includes proteins which differ from a naturallyoccurring human factor H (or biologically-active fragment thereof) inthat at least one or a few, but not limited to one or a few, amino acidshave been deleted (e.g., a truncated version of the protein, such as apeptide or fragment), inserted, inverted, substituted and/or derivatized(e.g., by glycosylation, phosphorylation, acetylation, myristoylation,prenylation, palmitation, amidation and/or addition ofglycosylphosphatidyl inositol). For example, a human factor H homologuemay have an amino acid sequence that is at least about 70% identical tothe amino acid sequence of a naturally occurring human factor H (e.g.,SEQ ID NO:3), for example at least about any of 75%, 76%, 77%, 78%, 79%,80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence ofa naturally occurring human factor H (e.g., SEQ ID NO:3). In certainembodiments, a homologue of human factor H (or a biologically-activefragment thereof) retains all the alternative complement pathwayinhibitory activity of human factor H (or a biologically-active fragmentthereof). In certain embodiments, the homologue of human factor H (or abiologically-active fragment thereof) retains at least about 50%, forexample, at least about any of 60%, 70%, 80%, 90%, or 95% of thecomplement inhibition activity of human factor H (or abiologically-active fragment thereof).

In certain embodiments, the biologically-active fragment of factor Hcomprises at least the first four N-terminal SCR domains of a humanfactor H, such as a factor H portion having an amino acid sequencecontaining at least amino acids 21 through 262 of the human factor H(SEQ ID NO:3). In certain embodiments, the biologically-active fragmentof factor H comprises at least the first four N-terminal SCR domains ofhuman factor H having an amino acid sequence that is at least about anyof 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to aminoacids 21 through 262 of the human factor H (SEQ ID NO:3).

In certain embodiments, the biologically-active fragment of factor Hcomprises at least the first five N-terminal SCR domains of a humanfactor H, such as a factor H portion having an amino acid sequencecontaining at least amino acids 21 through 320 of the human factor H(SEQ ID NO:3). In certain embodiments, the biologically-active fragmentof factor H comprises at least the first five N-terminal SCR domains ofhuman factor H having an amino acid sequence that is at least about anyof 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to aminoacids 21 through 320 of the human factor H (SEQ ID NO:3).

In certain embodiments, the biologically-active fragment of factor Hcomprises at least the first eight N-terminal SCR domains of a humanfactor H, such as a factor H portion having an amino acid sequencecontaining at least amino acids 21 through 509 of the human factor H(SEQ ID NO:3). In certain embodiments, the biologically-active fragmentof factor H comprises at least the first eight N-terminal SCR domains ofhuman factor H having an amino acid sequence that is at least about anyof 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to aminoacids 21 through 509 of the human factor H (SEQ ID NO:3).

In certain embodiments, the biologically-active fragment of factor Hcomprises at least the first eighteen N-terminal SCR domains of a humanfactor H, such as a factor H portion having an amino acid sequencecontaining at least amino acids 21 through 1106 of the human factor H(SEQ ID NO:3). In certain embodiments, the biologically-active fragmentof factor H comprises at least the first eighteen N-terminal SCR domainsof human factor H having an amino acid sequence that is at least aboutany of 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical toamino acids 21 through 1106 of the human factor H (SEQ ID NO:3).

As used herein, the term “membrane cofactor protein,” “MCP,” or “CD46”refers to a widely distributed C3b/C4b-binding cell surface glycoproteinwhich inhibits complement activation on host cells and serves as acofactor for the factor I-mediated cleavage of C3b and C4b, includinghomologues thereof. See T. J. Oglesby et al., J. Exp. Med. (1992)175:1547-1551. MCP belongs to a family known as the regulators ofcomplement activation (“RCA”). Family members share certain structuralfeatures, comprising varying numbers of short consensus repeat (SCR)domains, which are typically between 60 and 70 amino acids in length.MCP comprises four SCRs, a serine/threonine/proline-enriched region, anarea of undefined function, a transmembrane hydrophobic domain, acytoplasmic anchor and a cytoplasmic tail. It is understood that speciesand strain variations exist for the disclosed peptides, polypeptides,and proteins, and that human MCP or biologically-active fragmentsthereof encompasses all species and strain variations.

SEQ ID NO:19 represents the full-length human MCP amino acid sequence(see, e.g., UniProtKB/Swiss-Prot. Accession No. P15529). Amino acids1-34 correspond to the signal peptide, amino acids 35-343 correspond tothe extracellular domain, amino acids 344-366 correspond to thetransmembrane domain, and amino acids 367-392 correspond to thecytoplasmic domain. In the extracellular domain, amino acids 35-96correspond to SCR 1, amino acids 97-159 correspond to SCR 2, amino acids160-225 correspond to SCR 3, amino acids 226-285 correspond to SCR 4,and amino acids 302-326 correspond to the serine/threonine-rich domain.It is understood that species and strain variations exist for thedisclosed peptides, polypeptides, and proteins, and that MCP orbiologically-active fragments thereof encompasses all species and strainvariations. As used herein, the term “biologically-active” fragment ofMCP refers to any soluble fragment lacking both the cytoplasmic domainand the transmembrane domain, including fragments comprising, consistingessentially of or consisting of 1, 2, 3, or 4 SCR domains, with orwithout the serine/threonine-rich domain, having some or all thecomplement inhibitory activity of the full-length MCP protein.

In certain embodiments, the compositions suitable for inhibitingactivity of the alternative complement pathway in the kidneys comprise afusion protein comprising an anti-annexin A2 antibody or antigen-bindingfragment thereof fused to human membrane cofactor protein (MCP) (SEQ IDNOs: 19 and 20) or a biologically-active fragment thereof. In certainembodiments, the biologically-active fragment of human MCP comprises theextracellular domain of human MCP (amino acids 35-343 of SEQ ID NO:19),or SCRs 1 to 4 of human MCP (amino acids 35-285 of SEQ ID NO:19). Theanti-annexin A2 antibody or antigen-binding portion of the fusionprotein is responsible for delivering the composition to renal sites ofalternative complement activation by selectively binding to annexin A2expressed in the kidneys, while the MCP portion of the fusion protein isresponsible for inhibiting activity of the alternative complementpathway.

In certain embodiments, the fusion protein comprises an anti-annexin A2antibody or antigen-binding fragment thereof fused to full-length humanMCP. In certain embodiments, the fusion protein comprises ananti-annexin A2 antibody or antigen-binding fragment thereof fused to abiologically active fragment of full-length human MCP. In certainembodiments, the fusion protein comprises an anti-annexin A2 antibody orantigen-binding fragment thereof fused to a biologically active fragmentof full-length human MCP comprising the extracellular domain of humanMCP (amino acids 35-343 of SEQ ID NO:19). In certain embodiments, thefusion protein comprises an anti-annexin A2 antibody or antigen-bindingfragment thereof fused to a biologically active fragment of full-lengthhuman MCP comprising SCRs 1 to 4 (amino acids 35-285 of SEQ ID NO:19.

A homologue of a human MCP protein or a biologically-active fragmentthereof includes proteins which differ from a naturally occurring humanMCP (or biologically-active fragment thereof) in that at least one or afew, but not limited to one or a few, amino acids have been deleted(e.g., a truncated version of the protein, such as a peptide orfragment), inserted, inverted, substituted and/or derivatized (e.g., byglycosylation, phosphorylation, acetylation, myristoylation,prenylation, palmitation, amidation and/or addition ofglycosylphosphatidyl inositol). For example, a human MCP homologue mayhave an amino acid sequence that is at least about 70% identical to theamino acid sequence of a naturally occurring human MCP (e.g., SEQ IDNO:19), for example at least about any of 75%, 76%, 77%, 78%, 79%, 80%,81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of anaturally occurring human MCP (e.g., SEQ ID NO:19). In certainembodiments, a homologue of human MCP (or a biologically-active fragmentthereof) retains all the alternative complement pathway inhibitoryactivity of human MCP (or a biologically-active fragment thereof). Incertain embodiments, the homologue of human MCP (or abiologically-active fragment thereof) retains at least about 50%, forexample, at least about any of 60%, 70%, 80%, 90%, or 95% of thecomplement inhibition activity of human MCP (or a biologically-activefragment thereof).

As used herein, the term “CD59” refers to a membrane-bound 128 aminoacid glycoprotein that potently inhibits the membrane attack complex(MAC) of complement, including homologues thereof. CD59 acts by bindingto the C8 and/or C9 components of the MAC during assembly, ultimatelypreventing incorporation of the multiple copies of C9 required forcomplete formation of the osmolytic pore at the heart of the MAC. CD59is both N- and O-glycosylated. The N-glycosylation comprises primarilyof bi- or tri-antennary structures with and without lactosamine andouter arm fucose residues, with variable sialylation present at somesites. Like DAF, CD59 is anchored in the cell membrane by aglycosylphosphatidylinositol (“GPI”) anchor, which is attached to anasparagine at amino acid 102.

SEQ ID NO:21 represents the full-length human CD59 amino acid sequence(see, e.g., UniProtKB/Swiss-Prot. Accession No. P 13987). Amino acids1-25 correspond to the leader peptide, amino acids 26-102 correspond tothe mature protein, and amino acids 103-128 are removed aftertranslation. The GPI anchor is attached to CD59 at an asparagine atposition 102. It is understood that species and strain variations existfor the disclosed peptides, polypeptides, and proteins, and that CD59 orbiologically-active fragments thereof encompasses all species and strainvariations. As used herein, the term “biologically-active” fragment ofCD59 refers to any fragment of CD59 lacking a GPI anchor and/or theamino acid to which it is attached (i.e., Asn-102), including anyfragments of the full-length CD59 protein having some or all thecomplement inhibitory activity of the full-length CD59 protein.

In certain embodiments, the compositions suitable for inhibitingactivity of the alternative complement pathway in the kidneys comprise afusion protein comprising an anti-annexin A2 antibody or antigen-bindingfragment thereof fused to human CD59 (SEQ ID NOs:21 and 22) or abiologically-active fragment thereof. In certain embodiments, thebiologically-active fragment of human CD59 comprises the extracellulardomain of human CD59 (amino acids 26-102 of SEQ ID NO:21) lacking itsGPI anchor and/or the amino acid to which it is attached (i.e.,Asn-102). The anti-annexin A2 antibody or antigen-binding portion of thefusion protein is responsible for delivering the composition to renalsites of alternative complement activation by selectively binding toannexin A2 expressed in the kidneys, while the CD59 portion of thefusion protein is responsible for inhibiting activity of the alternativecomplement pathway.

In certain embodiments, the fusion protein comprises an anti-annexin A2antibody or antigen-binding fragment thereof fused to full-length humanCD59. In certain embodiments, the fusion protein comprises ananti-annexin A2 antibody or antigen-binding fragment thereof fused to abiologically active fragment of full-length human CD59. In certainembodiments, the fusion protein comprises an anti-annexin A2 antibody orantigen-binding fragment thereof fused to a biologically active fragmentof full-length human CD59 comprising the extracellular domain of humanCD59 (amino acids 26-102 of SEQ ID NO:21) lacking its GPI anchor and/orthe amino acid to which it is attached (i.e., Asn-102).

A homologue of a human CD59 protein or a biologically-active fragmentthereof includes proteins which differ from a naturally occurring humanCD59 (or biologically-active fragment thereof) in that at least one or afew, but not limited to one or a few, amino acids have been deleted(e.g., a truncated version of the protein, such as a peptide orfragment), inserted, inverted, substituted and/or derivatized (e.g., byglycosylation, phosphorylation, acetylation, myristoylation,prenylation, palmitation, amidation and/or addition ofglycosylphosphatidyl inositol). For example, a human MCP homologue mayhave an amino acid sequence that is at least about 70% identical to theamino acid sequence of a naturally occurring human CD59 (e.g., SEQ IDNO:21), for example at least about any of 75%, 76%, 77%, 78%, 79%, 80%,81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of anaturally occurring human CD59 (e.g., SEQ ID NO:21). In certainembodiments, a homologue of human CD59 (or a biologically-activefragment thereof) retains all the alternative complement pathwayinhibitory activity of human CD59 (or a biologically-active fragmentthereof). In certain embodiments, the homologue of human CD59 (or abiologically-active fragment thereof) retains at least about 50%, forexample, at least about any of 60%, 70%, 80%, 90%, or 95% of thecomplement inhibition activity of human CD59 (or a biologically-activefragment thereof).

As used herein, the term “complement receptor 1,” “CR1,” or “CD35”refers to a human gene encoding a protein of 2039 amino acids, with apredicted molecular weight of 220 kilodaltons (“kD”), includinghomologues thereof. The gene is expressed principally on erythrocytes,monocytes, neutrophils, and B cells, but is also present on some Tlymphocytes, mast cells, and glomerular podocytes. CR1 protein istypically expressed at between 100 and 1000 copies per cell. Thefull-length CR1 protein comprises a 42 amino acid signal peptide, anextracellular domain of 1930 amino acids, a 25 amino acid transmembranedomain, and a 43 amino acid C-terminal cytoplasmic domain. Theextracellular domain of CR1 has 25 potential N-glycosylation signalsequences, and comprises 30 short consensus (“SCR”) domains, also knownas complement control protein (CCP) repeats, or sushi domains, each 60to 70 amino acids long. The sequence homology between SCRs rangesbetween 60 to 99 percent. The 30 SCR domains are further grouped intofour longer regions termed long homologous repeats (“LHRs”), eachencoding approximately 45 kD segments of the CR1 protein, designatedLHR-A, -B, -C, and -D. The first three comprise seven SCR domains each,while LHR-D comprises 9 SCR domains. The active sites on theextracellular domain of CR1 protein include a C4b-binding site withlower affinity for C3b in SCRs 1 to 4 comprising amino acids 42-295, aC3b-binding site with lower affinity for C4b in SCRs 8 to 11 comprisingamino acids 490-745, a C3b-binding site with lower affinity for C4b inSCRs 15-18 comprising amino acids 940-1196, and a C1q-binding site inSCRs 22-28 comprising amino acids 1394-1842.

SEQ ID NO:23 represents the full-length human CR1 amino acid sequence(see, e.g., UniProtKB/Swiss-Prot. Accession No. P 17927). Amino acids1-41 correspond to the signal peptide, amino acids 42-2039 correspond tothe mature protein, comprising amino acids 42-1971, corresponding to theextracellular domain, amino acids 1972-1996, corresponding to thetransmembrane domain, and amino acids 1997-2039, corresponding to thecytoplasmic domain. In the extracellular domain, amino acids 42-101correspond to SCR 1, 102-163 correspond to SCR2, amino acids 164-234correspond to SCR3, amino acids 236-295 correspond to SCR4, amino acids295-355 correspond to SCR5, amino acids 356-418 correspond to SCR6,amino acids 419-489 correspond to SCR7, amino acids 491-551 correspondto SCR8, amino acids 552-613 correspond to SCR9, amino acids 614-684correspond to SCR10, amino acids 686-745 correspond to SCR11, aminoacids 745-805 correspond to SCR12, amino acids 806-868 correspond toSCR13, amino acids 869-939 correspond to SCR14, amino acids 941-1001correspond to SCR15, amino acids 1002-1063 correspond to SCR16, aminoacids 1064-1134 correspond to SCR17, amino acids 1136-1195 correspond toSCR18, amino acids 1195-1255 correspond to SCR 19, amino acids 1256-1318correspond to SCR 20, amino acids 1319-1389 correspond to SCR 21, aminoacids 1394-1454 correspond to SCR 22, amino acids 1455-1516 correspondto SCR 23, amino acids 1517-1587 correspond to SCR 24, amino acids1589-1648 correspond to SCR 25, amino acids 1648-1708 correspond to SCR26, amino acids 1709-1771 correspond to SCR 27, amino acids 1772-1842correspond to SCR 28, amino acids 1846-1906 correspond to SCR 29, aminoacids 1907-1967 correspond to SCR 30. It is understood that species andstrain variations exist for the disclosed peptides, polypeptides, andproteins, and that CR1 protein or biologically-active fragments thereofencompasses all species and strain variations. As used herein, the term“biologically-active” fragment of CR1 protein refers to refers to anysoluble fragment of CR1 lacking the transmembrane domain and thecytoplasmic domain, including fragments comprising, consistingessentially of or consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or30 SCR domains, including any fragments of the full-length CR1 proteinhaving some or all the complement inhibitory activity of the full-lengthCR1 protein.

A homologue of a human CR1 protein or a biologically-active fragmentthereof includes proteins which differ from a naturally occurring humanCR1 (or biologically-active fragment thereof) in that at least one or afew, but not limited to one or a few, amino acids have been deleted(e.g., a truncated version of the protein, such as a peptide orfragment), inserted, inverted, substituted and/or derivatized (e.g., byglycosylation, phosphorylation, acetylation, myristoylation,prenylation, palmitation, amidation and/or addition ofglycosylphosphatidyl inositol). For example, a human CR1 homologue mayhave an amino acid sequence that is at least about 70% identical to theamino acid sequence of a naturally occurring human CR1 (e.g., SEQ IDNO:23), for example at least about any of 75%, 76%, 77%, 78%, 79%, 80%,81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of anaturally occurring human CR1 (e.g., SEQ ID NO:23). In certainembodiments, a homologue of human CR1 (or a biologically-active fragmentthereof) retains all the alternative complement pathway inhibitoryactivity of human CR1 (or a biologically-active fragment thereof). Incertain embodiments, the homologue of human CR1 (or abiologically-active fragment thereof) retains at least about 50%, forexample, at least about any of 60%, 70%, 80%, 90%, or 95% of thecomplement inhibition activity of human CR1 (or a biologically-activefragment thereof).

In certain embodiments, the compositions suitable for inhibitingactivity of the alternative complement pathway in the kidneys comprise afusion protein comprising an anti-annexin A2 antibody or antigen-bindingfragment thereof fused to human CR1 (SEQ ID NOs:23 and 24) or abiologically-active fragment thereof. In certain embodiments, thebiologically-active fragment of human CR1 comprises the completeextracellular domain of human CR1 (SCRs 1 to 30), SCRs 1 to 4, SCRs 1 to11, or SCRs 1 to 18. The anti-annexin A2 antibody or antigen-bindingportion of the fusion protein is responsible for delivering thecomposition to renal sites of alternative complement activation byselectively binding to annexin A2 expressed in the kidneys, while theCR1 portion of the fusion protein is responsible for inhibiting activityof the alternative complement pathway.

In certain embodiments, the fusion protein comprises an anti-annexin A2antibody or antigen-binding fragment thereof fused to full-length humanCR1. In certain embodiments, the fusion protein comprises ananti-annexin A2 antibody or antigen-binding fragment thereof fused to abiologically active fragment of full-length human CR1 comprising thecomplete extracellular domain of human CR1 (SCRs 1 to 30)(amino acids42-1971 of SEQ ID NO:23). In certain embodiments, the fusion proteincomprises an anti-annexin A2 antibody or antigen-binding fragmentthereof fused to a biologically active fragment of full-length human CR1comprising SCRs 1 to 4 (amino acids 42-295 of SEQ ID NO:23). In certainembodiments, the fusion protein comprises an anti-annexin A2 antibody orantigen-binding fragment thereof fused to a biologically active fragmentof full-length human CR1 comprising SCRs 1 to 11 (amino acids 42-745 ofSEQ ID NO:23). In certain embodiments, the fusion protein comprises ananti-annexin A2 antibody or antigen-binding fragment thereof fused to abiologically active fragment of full-length human CR1 comprising SCRs 1to 18 (amino acids 42-1195 of SEQ ID NO:23).

As used herein, the term “mouse complement receptor 1-relatedgene/protein y” or “Crry” refers to a membrane-bound mouse glycoproteinthat regulates complement activation, including homologues thereof. Crryregulates complement activation by serving as a cofactor for complementfactor I, a serine protease which cleaves C3b and C4b deposited on hosttissue. Crry also acts as a decay-accelerating factor, preventing theformation of C4b2a and C3bBb, the amplification convertases of thecomplement cascade.

SEQ ID NO:25 represents the full-length mouse Crry protein amino acidsequence. Amino acids 1-40 correspond to the leader peptide, amino acids41-483 correspond to the mature protein, comprising amino acids 41-405,corresponding to the extracellular domain, amino acids 406-426,corresponding to the transmembrane domain, and amino acids 427-483,corresponding to the cytoplasmic domain. In the extracellular domain,amino acids 83-143 correspond to SCR 1, 144-205 correspond to SCR2,amino acids 206-276 correspond to SCR3, amino acids 277-338 correspondto SCR4, and amino acids 339-400 correspond to SCR5. It is understoodthat species and strain variations exist for the disclosed peptides,polypeptides, and proteins, and that mouse Crry protein orbiologically-active fragments thereof encompasses all species and strainvariations. As used herein, the term “biologically-active” fragment ofmouse Crry protein refers to refers to any soluble fragment of mouseCrry lacking the transmembrane domain and the cytoplasmic domain,including fragments comprising, consisting essentially of or consistingof 1, 2, 3, 4, or 5 SCR domains, including any fragments of thefull-length mouse Crry protein having some or all the complementinhibitory activity of the full-length Crry protein.

A homologue of a mouse Crry protein or a biologically-active fragmentthereof includes proteins which differ from a naturally occurring mouseCrry protein (or biologically-active fragment thereof) in that at leastone or a few, but not limited to one or a few, amino acids have beendeleted (e.g., a truncated version of the protein, such as a peptide orfragment), inserted, inverted, substituted and/or derivatized (e.g., byglycosylation, phosphorylation, acetylation, myristoylation,prenylation, palmitation, amidation and/or addition ofglycosylphosphatidyl inositol). For example, a mouse Crry proteinhomologue may have an amino acid sequence that is at least about 70%identical to the amino acid sequence of a naturally occurring mouse Crryprotein (e.g., SEQ ID NO:25), for example at least about any of 75%,76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to theamino acid sequence of a naturally occurring mouse Crry protein (e.g.,SEQ ID NO:25). In certain embodiments, a homologue of mouse Crry protein(or a biologically-active fragment thereof) retains all the alternativecomplement pathway inhibitory activity of mouse Crry protein (or abiologically-active fragment thereof). In certain embodiments, thehomologue of mouse Crry protein (or a biologically-active fragmentthereof) retains at least about 50%, for example, at least about any of60%, 70%, 80%, 90%, or 95% of the complement inhibition activity ofmouse Crry protein (or a biologically-active fragment thereof).

In certain embodiments, the compositions suitable for inhibitingactivity of the alternative complement pathway in the kidneys comprise afusion protein comprising an anti-annexin A2 antibody or antigen-bindingfragment thereof fused to mouse Crry (SEQ ID NOs:25 and 26) or abiologically-active fragment thereof. In certain embodiments, thebiologically-active fragment of mouse Crry comprises the completeextracellular domain of mature mouse Crry (amino acids 41-405 of SEQ IDNO:25) or short consensus repeat sequences 1 to 5 (SCRs 1 to 5)(aminoacids 83-400 of SEQ ID NO:25). The anti-annexin A2 antibody orantigen-binding portion of the fusion protein is responsible fordelivering the composition to renal sites of alternative complementactivation by selectively binding to annexin A2 expressed in thekidneys, while the mouse Crry portion of the fusion protein isresponsible for inhibiting activity of the alternative complementpathway.

In certain embodiments, the fusion protein comprises an anti-annexin A2antibody or antigen-binding fragment thereof fused to full-length mouseCrry. In certain embodiments, the fusion protein comprises ananti-annexin A2 antibody or antigen-binding fragment thereof fused to abiologically active fragment of full-length mouse Crry. In certainembodiments, the fusion protein comprises an anti-annexin A2 antibody orantigen-binding fragment thereof fused to a biologically active fragmentof full-length mouse Crry comprising the complete extracellular domainof mature mouse Crry (amino acids 41-405 of SEQ ID NO:25). In certainembodiments, the fusion protein comprises an anti-annexin A2 antibody orantigen-binding fragment thereof fused to a biologically active fragmentof full-length mouse Crry comprising SCRs 1 to 5 (amino acids 83-400 ofSEQ ID NO:25).

In some embodiments, the fusion protein comprising an anti-annexin A2antibody or antigen-binding fragment thereof and a DAF, factor H, MCP,CD59, CR1, or mouse Crry protein or a biologically-active fragmentthereof also includes an amino acid linker sequence linking theanti-annexin A2 portion and the complement inhibitor portion (e.g., theDAF, factor H, MCP, CD59, CR1, or mouse Crry protein portion). Examplesof linker sequences are known in the art, and include, for example,(Gly₄Ser), (Gly₄Ser)₂, (Gly₄Ser)₃, (Gly₃Ser)₄, (SerGly₄), (SerGly₄)₂,(SerGly₄)₃, and (SerGly₄)₄. Linking sequences can also comprise“natural” linking sequences found between different domains ofcomplement factors. For example, VSVFPLE (SEQ ID NO:30), the linkingsequence between the first two N-terminal short consensus repeat domainsof human CR2, can be used. In some embodiments, the linking sequencebetween the fourth and the fifth N-terminal short consensus repeatdomains of human CR2 (EEIF) is used.

As used herein, the term “specifically binds to” or “selectively bindsto” refers to the specific binding of one protein to another (e.g., anantibody or antigen-binding fragment thereof to an antigen, or areceptor to a ligand), wherein the level of binding, as measured by anystandard assay (e.g., an immunoassay), is statistically significantlyhigher than the background control for the assay. For example, whenperforming an immunoassay, controls typically include a reaction well ortube that contains an antibody or antigen-binding fragment thereof alone(i.e., in the absence of antigen), wherein an amount of reactivity(e.g., non-specific binding to the well) by the antibody orantigen-binding fragment thereof in the absence of the antigen isconsidered to be background. Binding can be measured using a variety ofmethods standard in the art, including, but not limited to: Westernblot, immunoblot, enzyme-linked immunosorbant assay (“ELISA”),radioimmunoassay (“RIA”), immunoprecipitation, surface plasmonresonance, chemiluminescence, fluorescent polarization, phosphorescence,immunohistochemical analysis, matrix-assisted laserdesorption/ionization time-of-flight (“MALDI-TOF”) mass spectrometry,microcytometry, microarray, microscopy, fluorescence activated cellsorting (“FACS”), and flow cytometry.

As used herein, the term “anti-annexin A2 antibody or antigen-bindingfragment thereof” refers to an antibody that specifically or selectivelybinds to annexin A2, or a fragment of such an antibody that retains theability to specifically or selectively bind to annexin A2, preferablywithout interfering with the binding of endogenous native factor H.Antibodies contain immunoglobulin (Ig) domains and are members of the Igsuperfamily of proteins. Generally, an antibody molecule comprises twotypes of chains: a heavy or H chain, and a light or L chain. The lightchain contains a variable domain (V_(L)) and a constant domain (C_(L)),while the heavy chain contains a variable domain (V_(H)) and threeconstant domains (C_(H)1, C_(H)2, and C_(H)3), with the C_(H)1 andC_(H)2 domains separated by a hinge region. The distinctivecharacteristics of each isotype are defined by sequences in the constantdomain of the immunoglobulin. Each antibody molecule typically containstwo H chains and two L chains. The two H chains are linked together bydisulfide bonds and each H chain is linked to an L chain by a disulfidebond. There are only two types of L chains referred to as lambda (λ) andkappa (κ) chains. In contrast, there are five major H chain classes,referred to as isotypes. The five classes include IgM (i), IgD (δ), IgG(λ), IgA (α), and IgE (or ε). Human immunoglobulin molecules comprisenine isotypes: IgM, IgD, IgE, four subclasses of IgG including IgG₁(γ₁), IgG₂ (γ₂), IgG₃ (γ₃) and IgG₄ (γ₄), and two subclasses of IgAincluding IgA₁ (α₁) and IgA₂ (α₂).

Together, one H chain and one L chain form an arm of an immunoglobulinmolecule having an immunoglobulin variable region. A completeimmunoglobulin molecule comprises two di-sulfide linked arms. Thus, eacharm of a whole immunoglobulin comprises a V_(H+L) region, and a C_(H+L)region. As used herein, the variable region or V region refers to aV_(H+L) region (also known as an Fv fragment), a V_(L) region, or aV_(H) region of an Ig protein. Also as used herein, the term constantregion or C region refers to a C_(H+L) region, a C_(L) region or a C_(H)region.

Limited digestion of an Ig protein with different proteases produces anumber of fragments, only some of which retain the capacity to bindantigen. The antigen-binding fragments are referred to as Fab, Fab′, orF(ab′)₂ fragments. A fragment lacking the ability to bind to antigen isreferred to as an Fc fragment. An Fab fragment comprises one arm of animmunoglobulin molecule containing an L chain (V_(L)+C_(L) domains)paired with the V_(H) region and the C_(H)1 region. An Fab′ fragmentcorresponds to an Fab fragment with part of the hinge region attached tothe C_(H)1 domain. An F(ab′)₂ fragment corresponds to two Fab′ fragmentsthat are normally covalently linked to each other through a disulfidebond, typically in the hinge region.

The anti-annexin A2 antibodies or antigen-binding fragments thereof mayalso be “chimeric” antibodies in which a portion of the heavy and/orlight chain is identical with or homologous to corresponding sequencesin antibodies derived from a particular species (e.g., human or mouseand the like) or belonging to a particular antibody class or subclass(e.g., IgG₁ and the like), while the remainder of the chain(s) is(are)identical with or homologous to corresponding sequences in antibodiesderived from another species or belonging to another antibody class orsubclass, as well as fragments of such antibodies, so long as theyexhibit the desired biological activity. See U.S. Pat. No. 4,816,567;Morrison et al., Proc. Nat'l Acad. Sci. USA, 81:6851-55 (1984). Chimericantibodies of interest herein may include, for example, those comprisingFc domains from other immunoglobulin subtypes having shorter or longercirculating plasma half lives than the corresponding non-chimericanti-annexin A2 antibody.

Anti-annexin A2 antibodies or antigen-binding fragments thereof may alsobe humanized antibodies. Humanized antibodies are molecules having anantigen-binding site derived from an immunoglobulin from a non-humanspecies, the remaining immunoglobulin-derived parts of the moleculebeing derived from a human immunoglobulin, in order to reduceimmunogenicity of the protein. The antigen-binding site may compriseeither complete variable regions fused onto human constant domains oronly the complementarity determining regions (CDRs) grafted ontoappropriate human framework regions in the variable domains. Humanizedantibodies can be produced, for example, by modeling the antibodyvariable domains and producing the antibodies using genetic engineeringtechniques, such as CDR grafting. A description of various techniquesfor the production of humanized antibodies is found, for example, inMorrison et al., (1984) Proc. Nat'l Acad. Sci. USA 81:6851-55; Whittleet al., (1987) Prot. Eng. 1:499-505; Co et al., (1990) J. Immunol.148:1149-1154; Co et al., (1992) Proc. Nat'l Acad. Sci. USA88:2869-2873; Carter et al., (1992) Proc. Nat'l Acad. Sci. USA89:4285-4289; Routledge et al., (1991) Eur. J. Immunol. 21:2717-2725 andPCT Patent Publication Nos. WO 91/09967; WO 91/09968 and WO 92/113831.

Whole antibodies as described herein can be polyclonal or monoclonal.Alternatively, functional equivalents of whole antibodies, such asantigen-binding fragments in which one or more antibody domains aretruncated or absent (e.g., Fv, Fab, Fab′, or F(ab)₂ fragments), as wellas genetically-engineered antibodies or antigen-binding fragmentsthereof, including single chain antibodies, humanized antibodies(discussed above), antibodies that can bind to more than one epitope(e.g., bi-specific antibodies), or antibodies that can bind to one ormore different antigens (e.g., bi- or multi-specific antibodies), mayalso be used as targeting groups.

Methods of producing polyclonal antibodies that specifically orselectively bind to a particular antigen (i.e., annexin A2) are known inthe art. Generally, in the production of an antibody, a suitableexperimental animal, such as, for example, but not limited to, a rabbit,a sheep, a hamster, a guinea pig, a mouse, a rat, or a chicken, isexposed to an antigen against which an antibody is desired (i.e.,annexin A2). Typically, an animal is immunized with an effective amountof antigen that is injected into the animal. An effective amount ofantigen refers to an amount needed to induce antibody production by theanimal. The animal's immune system is then allowed to respond over apre-determined period of time. The immunization process can be repeateduntil the immune system is found to be producing antibodies to theantigen. In order to obtain polyclonal antibodies specific for theantigen, serum is collected from the animal that contains the desiredantibodies (or in the case of a chicken, antibody can be collected fromthe eggs). Such serum is useful as a reagent. Polyclonal antibodies canbe further purified from the serum (or eggs) by, for example, treatingthe serum with ammonium sulfate to precipitate the antibodies.

Methods of producing monoclonal antibodies that specifically orselectively bind to a particular antigen (i.e., annexin A2) are known inthe art. For example, monoclonal antibodies may be produced according tothe methodology of Kohler and Milstein (Nature (1975) 256:495-497). Forexample, B lymphocytes are recovered from the spleen (or any suitabletissue) of an immunized animal and then fused with myeloma cells toobtain a population of hybridoma cells capable of continual growth insuitable culture medium. Hybridomas producing the desired antibody areselected by testing the ability of the antibody produced by thehybridoma to bind to the desired antigen, for example in anenzyme-linked immunosorbent assay or other routine method known in theart.

A preferred method to produce antibodies described herein includes (a)administering to an animal an effective amount of a protein or peptide(e.g., an annexin A2 protein or peptide including domains thereof) toproduce the antibodies and (b) recovering the antibodies. In anothermethod, antibodies described herein are produced recombinantly. Forexample, once a cell line, for example a hybridoma, expressing anantibody useful for the compositions or methods described herein hasbeen obtained, it is possible to clone therefrom the cDNA and toidentify the variable region genes encoding the desired antibody,including the sequences encoding the CDRs. From there, antibodies andantigen-binding fragments as described herein may be obtained bypreparing one or more replicable expression vectors containing at leastthe DNA sequence encoding the variable domain of the antibody heavy orlight chain and optionally other DNA sequences encoding remainingportions of the heavy and/or light chains as desired, and transformingor transfecting an appropriate host cell, in which production of theantibody will occur. Suitable expression hosts include bacteria, (forexample, an E. coli strain), fungi, (in particular yeasts, e.g., membersof the genera Pichia, Saccharomyces, or Kluyveromyces) and mammaliancell lines, e.g., a non-producing myeloma cell line, such as a mouse NSOline, or CHO cells. In order to obtain efficient transcription andtranslation, the DNA sequence in each vector should include appropriateregulatory sequences, particularly a promoter and leader sequenceoperably linked to the variable domain sequence. Particular methods forproducing antibodies in this way are generally well known and routinelyused. For example, basic molecular biology procedures are described byManiatis et al. (Molecular Cloning, Cold Spring Harbor Laboratory, NewYork, 1989); DNA sequencing can be performed as described in Sanger etal. (Proc. Nat'l Acad. Sci. USA (1977) 74:5463) and the AmershamInternational plc sequencing handbook; and site directed mutagenesis canbe carried out according to the method of Kramer et al. (Nucl. AcidsRes. (1984) 12:9441), and the Anglian Biotechnology Ltd. handbook.Additionally, there are numerous publications, including patentspecifications, describing techniques suitable for the preparation ofantibodies by manipulation of DNA, creation of expression vectors andtransformation of appropriate cells, for example as reviewed byMountain, A. and Adair, J. R., in BIOTECHNOLOGY AND GENETIC ENGINEERINGREVIEWS (ed. Tombs, M P, 10, Chapter 1, 1992, Intercept, Andover, UK).

In certain embodiments, the factor H portion of the fusion proteincompositions suitable for inhibiting activity of the alternativecomplement pathway in the kidneys comprises homologues of human factor Hprotein (SEQ ID NO:3) or biologically-active fragments thereof. Ahomologue of a human factor H protein or biologically-active fragmentthereof includes proteins which differ from a naturally occurring humanfactor H protein (or from a biologically-active fragment thereof) inthat at least one or a few, but not limited to one or a few, amino acidshave been deleted (e.g., a truncated version of the protein, such as apeptide or fragment), inserted, inverted, substituted and/or derivatized(e.g., by glycosylation, phosphorylation, acetylation, myristoylation,prenylation, palmitation, amidation and/or addition ofglycosylphosphatidyl inositol). For example, a factor H homologue orbiologically-active fragments thereof may have an amino acid sequencethat is at least about 70% identical to the amino acid sequence of anaturally occurring human factor H or biologically-active fragmentsthereof (e.g., SEQ ID NO:3), for example at least about any of 75%, 76%,77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the aminoacid sequence of a naturally occurring human factor H (e.g., SEQ IDNO:3). In certain embodiments, a homologue of factor H (orbiologically-active fragments thereof) retains all the biologicalactivities of human factor H (or biologically-active fragments thereof),i.e., the biologically-active fragments of factor H have one or more ofthe following properties: (1) binding to C-reactive protein (CRP), (2)binding to C3b, (3) binding to heparin, (4) binding to sialic acid, (5)binding to endothelial cell surfaces, (6) binding to cellular integrinreceptor, (7) binding to pathogens, (8) C3b co-factor activity, (9) C3bdecay-acceleration activity, and (10) inhibiting the alternativecomplement pathway.

In certain embodiments, the factor H portion of the fusion proteincompositions suitable for inhibiting activity of the alternativecomplement pathway in the kidneys comprises full-length factor H. Incertain embodiments, the factor H portion of the fusion proteincompositions suitable for inhibiting activity of the alternativecomplement pathway in the kidneys comprises a biologically-activefragment of factor H. In certain embodiments, the biologically-activefragment of factor H comprises the first four N-terminal SCR domains offactor H. In certain embodiments, the biologically-active fragment offactor H comprises the first five N-terminal SCR domains of factor H. Incertain embodiments, the biologically-active fragment of factor Hcomprises the first six N-terminal SCR domains of factor H. In certainembodiments, the biologically-active fragment of factor H comprises thefirst eight N-terminal SCR domains of factor H. In certain embodiments,the biologically-active fragment of factor H comprises the firsteighteen N-terminal SCR domains of factor H. In certain embodiments, thebiologically-active fragment of factor H comprises SCRs 1 to 4 of factorH. In certain embodiments, the biologically-active fragment of factor Hcomprises SCRs 1 to 8 of factor H. In certain embodiments, thebiologically-active fragment of factor H comprises SCRs 1 to 18 offactor H. In certain embodiments, the factor H portion of the fusionprotein compositions comprises (and in certain embodiments consists ofor consists essentially of) at least the first four N-terminal SCRdomains of factor H, including for example, at least any of the first 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 or more N-terminal SCRdomains of factor H.

In certain embodiments, the factor H or biologically-active fragmentthereof portion of the fusion protein composition is or is derived froma wildtype factor H. In certain embodiments, the factor H orbiologically-active fragment thereof portion of the fusion proteincomposition is or is derived from a protective variant of factor H.

In certain embodiments, the factor H or biologically-active fragmentthereof portion of the fusion protein composition lacks a heparinbinding site. This can be achieved, for example, by mutation of theheparin binding site on factor H, or by selecting biologically-activefactor H fragments that do not contain a heparin binding site. Incertain embodiments, the factor H or biologically-active fragmentthereof portion of the fusion protein composition comprises factor H orbiologically-active fragments thereof having a polymorphism that isprotective to age-related macular degeneration. See Hageman et al.,Proc. Nat'l Acad. Sci. USA 102(20):7227.

In certain embodiments, the compositions are suitable for stimulatingactivity of the alternative complement pathway. In certain embodiments,activity of the alternative complement pathway is stimulated in thekidneys. In certain embodiments, the compositions suitable forstimulating activity of the complement pathway comprise abiologically-active fragment of factor H lacking the complementregulatory domain present in SCRs 1 to 4 of full-length factor H. Suchbiologically-active fragments of factor H retain the ability to bindcomplement protein C3 breakdown product C3d but lack the ability toinhibit alternative complement activity. In certain embodiments, thebiologically-active fragment of factor H comprises (and in certainembodiments consists of or consists essentially of) at least the lastsixteen C-terminal SCR domains of factor H, including for example, atleast any of the last 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3,or 2 C-terminal SCR domains of factor H. In certain embodiments, thebiologically-active fragment of factor H comprises SCRs 19 to 20 offactor H (SEQ ID NO:8).

Also encompassed herein are variants any of the compositions describedherein, for example, of annexin A2 or biologically-active fragmentsthereof, fusion proteins comprising anti-annexin A2 antibodies orantigen-binding fragments thereof fused to DAF, factor H, MCP, CD59,CR1, mouse Crry protein or biologically-active fragments thereof, orDAF, factor H, MCP, CD59, CR1, mouse Crry protein molecules orbiologically-active fragments thereof. Such variants may be: (i)variants in which one or more of the amino acid residues of the annexinA2 or biologically-active fragments thereof, fusion proteins comprisinganti-annexin A2 antibodies or antigen-binding fragments thereof fused toDAF, factor H, MCP, CD59, CR1, mouse Crry protein or biologically-activefragments thereof, or factor H molecules or biologically-activefragments thereof are substituted with a conserved or non-conservedamino acid residue (preferably a conserved amino acid residue) and suchsubstituted amino acid residue may or may not be one encoded by thegenetic code; or (ii) variants in which one or more of the amino acidresidues in the annexin A2 or biologically-active fragments thereof,fusion proteins comprising anti-annexin A2 antibodies or antigen-bindingfragments thereof fused to DAF, factor H, MCP, CD59, CR1, mouse Crryprotein or biologically-active fragments thereof, or factor H moleculesor biologically-active fragments thereof includes a substituent group,or (iii) one in which the annexin A2 or biologically-active fragmentsthereof, fusion proteins comprising anti-annexin A2 antibodies orantigen-binding fragments thereof fused to DAF, factor H, MCP, CD59,CR1, mouse Crry protein or biologically-active fragments thereof, orfactor H molecules or biologically-active fragments thereof is fusedwith another compound, such as a compound to increase the half-life ofthe composition (for example, polyethylene glycol), or (iv) one in whichadditional amino acids are fused to the annexin A2 orbiologically-active fragments thereof, fusion proteins comprisinganti-annexin A2 antibodies or antigen-binding fragments thereof fused toDAF, factor H, MCP, CD59, CR1, mouse Crry protein or biologically-activefragments thereof, or factor H molecules or biologically-activefragments thereof, such as a leader or secretory sequence or a sequencewhich is employed for purification of the composition, or (v) one inwhich the annexin A2 or biologically-active fragments thereof, fusionproteins comprising anti-annexin A2 antibodies or antigen-bindingfragments thereof fused to DAF, factor H, MCP, CD59, CR1, mouse Crryprotein or biologically-active fragments thereof, or factor H moleculesor biologically-active fragments thereof is fused with a largerpolypeptide, i.e., human albumin, an antibody or Fc, for increasedduration of effect. Such variants are deemed to be within the scope ofthose skilled in the art from the teachings herein.

In certain embodiments, the variant of the annexin A2 orbiologically-active fragments thereof, fusion proteins comprisinganti-annexin A2 antibodies or antigen-binding fragments thereof fused toDAF, factor H, MCP, CD59, CR1, mouse Crry protein or biologically-activefragments thereof, or factor H molecules or biologically-activefragments thereof contains conservative amino acid substitutions(defined further below) made at one or more predicted, preferablynonessential amino acid residues. A nonessential amino acid residue is aresidue that can be altered from the wild-type sequence of a proteinwithout altering the biological activity, whereas an essential aminoacid residue is required for biological activity. A conservative aminoacid substitution is one in which the amino acid residue is replacedwith an amino acid residue having a similar side chain. Families ofamino acid residues having similar side chains have been defined in theart. These families include amino acids with basic side chains (e.g.,lysine, arginine, histidine), acidic side chains (e.g., aspartic acid,glutamic acid), uncharged polar side chains (e.g., glycine, asparagine,glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains(e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine,methionine, tryptophan), beta-branched side chains (e.g., threonine,valine, isoleucine) and aromatic side chains (e.g., tyrosine,phenylalanine, tryptophan, histidine).

Amino acid substitutions can be introduced in annexin A2 orbiologically-active fragments thereof, fusion proteins comprisinganti-annexin A2 antibodies or antigen-binding fragments thereof fused toDAF, factor H, MCP, CD59, CR1, mouse Crry protein or biologically-activefragments thereof, or factor H molecules or biologically-activefragments thereof to improve the functionality of the molecule. Forexample, amino acid substitutions can be introduced into annexin A2 orbiologically-active fragments thereof to increase binding affinity ofannexin A2 for its ligand(s), to increase binding specificity of annexinA2 for its ligand(s), to increase dimerization or multimerization ofannexin A2 or biologically-active fragments thereof, and to improvepharmacokinetics of the annexin A2 or biologically-active fragmentsthereof. Similarly, amino acid substitutions can be introduced intofactor H or biologically-active fragments thereof to increase thefunctionality of the composition comprising anti-annexin A2 antibody orantigen-binding fragments thereof fused to factor H orbiologically-active fragments thereof and to improve thepharmacokinetics of the fusion proteins.

In certain embodiments, any of the stimulatory or inhibitorycompositions described herein (such as the annexin A2 protein orbiologically-active fragments thereof, the fusion proteins comprisinganti-annexin A2 antibodies or antigen-binding fragments thereof fused toDAF, factor H, MCP, CD59, CR1, mouse Crry protein or biologically-activefragments thereof, or factor H molecules or biologically-activefragments thereof) is fused with another compound, such as a compound toincrease the half-life of the polypeptide and/or to reduce potentialimmunogenicity of the polypeptide (for example, polyethylene glycol, orPEG). PEG can be used to impart water solubility, size, slow rate ofkidney clearance, and reduced immunogenicity to the fusion protein. See,e.g., U.S. Pat. No. 6,214,966. In the case of PEGylations, the fusion ofany of the compositions described herein (such as the annexin A2 proteinor biologically-active fragments thereof, the fusion proteins comprisinganti-annexin A2 antibodies or antigen-binding fragments thereof fused toDAF, factor H, MCP, CD59, CR1, mouse Crry protein or biologically-activefragments thereof, or factor H molecules or biologically-activefragments thereof) to PEG can be accomplished by any means known to oneskilled in the art. For example, PEGylation can be accomplished by firstintroducing a cysteine mutation into the desired composition, followedby site-specific derivatization with PEG-maleimide. The cysteine can beadded to the C-terminus of the composition. See, e.g., Tsutsumi et al.,(2000) Proc. Nat'l Acad. Sci. USA 97(15):8548-8553. Another modificationwhich can be made to any of the compositions described herein (such asthe annexin A2 protein or biologically-active fragments thereof, fusionproteins comprising anti-annexin A2 antibodies or antigen-bindingfragments thereof fused to DAF, factor H, MCP, CD59, CR1, mouse Crryprotein or biologically-active fragments thereof, or factor H moleculesor biologically-active fragments thereof) involves biotinylation. Incertain instances, it may be useful to have the composition biotinylatedso that it can readily react with streptavidin. Methods forbiotinylation of proteins are well known in the art. Additionally,chondroitin sulfate can be linked with any of the compositions describedherein.

Any of the compositions described herein (such as the annexin A2 proteinor biologically-active fragments thereof, the fusion proteins comprisinganti-annexin A2 antibodies or antigen-binding fragments thereof fused toDAF, factor H, MCP, CD59, CR1, mouse Crry protein or biologically-activefragments thereof, or factor H molecules or biologically-activefragments thereof) may also be modified to include an immunologicallyactive domain, such as an antibody epitope or other tag, to facilitatetargeting or purification of the polypeptide. The use of 6×His and GST(glutathione-S-transferase) as tags is well known. Inclusion of acleavage site at or near the fusion junction will facilitate removal ofthe extraneous polypeptide after purification. Other amino acidsequences that may be included in the compositions described hereininclude functional domains, such as active sites from enzymes such as ahydrolase, glycosylation domains, and cellular targeting signals.

Variants of the compositions described herein include polypeptideshaving an amino acid sequence sufficiently similar to the amino acidsequence of the annexin A2 protein or biologically-active fragmentsthereof, the fusion proteins comprising anti-annexin A2 antibodies orantigen-binding fragments thereof fused to DAF, factor H, MCP, CD59,CR1, mouse Crry protein or biologically-active fragments thereof, orfactor H molecules or biologically-active fragments thereof. The termsufficiently similar means a first amino acid sequence that contains asufficient or minimum number of identical or equivalent amino acidresidues relative to a second amino acid sequence such that the firstand second amino acid sequences have a common structural domain and/orcommon functional activity. For example, amino acid sequences thatcontain a common structural domain that is at least about 45%,preferably about 75% through 98%, identical are defined herein assufficiently similar. Variants include variants of fusion proteinsencoded by a polynucleotide that hybridizes to a polynucleotide asdescribed herein or a complement thereof under stringent conditions.Stringent hybridization conditions are known to those skilled in theart, and are described, for example, in references such as MOLECULARCLONING, by Sambrook and Russell. 3d ed., Cold Spring Harbor Press, ColdSpring Harbor, N.Y. (2001). Such variants generally retain thefunctional activity of the compositions described herein. Libraries offragments of the polynucleotides can be used to generate a variegatedpopulation of fragments for screening and subsequent selection. Forexample, a library of fragments can be generated by treating adouble-stranded PCR fragment of a polynucleotide with a nuclease underconditions wherein nicking occurs only about once per molecule,denaturing the double-stranded DNA, renaturing the DNA to formdouble-stranded DNA which can include sense/antisense pairs fromdifferent nicked products, removing single-stranded portions fromreformed duplexes by treatment with S1 nuclease, and ligating theresulting fragment library into an expression vector. By this method,one can derive an expression library that encodes N-terminal andinternal fragments of various sizes of the compositions describedherein.

In certain embodiments, any of the compositions described herein,particularly the annexin A2 protein or biologically-active fragmentsthereof, the fusion proteins comprising anti-annexin A2 antibodies orantigen-binding fragments thereof fused to DAF, factor H, MCP, CD59,CR1, mouse Crry protein or biologically-active fragments thereof, orfactor H molecules or biologically-active fragments thereof, are fusedat their N-terminus to a signal peptide. Such signal peptides guidesecretion of the molecules, facilitating purification from culturemedium. Suitable signal peptides include, for example, the signalpeptide of the CD5 protein (such as signal peptide of the human CD5protein MPMGSLQPLATLYLLGMLVAS, SEQ ID NO: 11). In certain embodiments,the signal peptide of the CR2 protein is used. For example, in certainembodiments, the signal peptide of the human CR2 protein(MGAAGLLGVFLALVAPG, SEQ ID NO:13 or MGAAGLLGVFLALVAPGVLG, SEQ ID NO:15)is used.

Any of the compositions described herein may be made by chemicalsynthesis methods, or by recombinant methods, for example, by preparinga polynucleotide encoding human annexin A2 or biologically-activefragments thereof, or polynucleotides encoding an anti-annexin A2antibody (light and heavy chains) and DAF, factor H, MCP, CD59, CR1,mouse Crry protein molecules or biologically-active fragments thereof(with or without a linker sequence), and introducing the resultingpolynucleotide molecule(s) into a vector for transfecting host cellsthat are capable of expressing the molecule. Chemical synthesis,especially solid phase synthesis, is preferred for short peptides orthose containing unnatural or unusual amino acids such as D-tyrosine,ornithine, and the like. Recombinant procedures are preferred for longerpolypeptides. Recombinant preparations of any of the compositionsdescribed herein can be isolated in vitro by routine proteinpurification methods. The compositions described and provided herein canalso be provided in situ by introduction of a gene therapy system to thetissue of interest which then expresses the desired composition.

Recombinant DNA techniques for making the compositions described andprovided herein involve, in simplified form, taking a polynucleotideencoding the desired composition (e.g., the annexin A2 protein orbiologically-active fragments thereof, the fusion proteins comprisinganti-annexin A2 antibodies or antigen-binding fragments thereof fused toDAF, factor H, MCP, CD59, CR1, mouse Crry protein or biologically-activefragments thereof, or factor H molecules or biologically-activefragments thereof), inserting it into an appropriate vector, insertingthe vector into an appropriate host cell, and recovering or otherwiseisolating the protein produced thereby.

Provided herein are polynucleotides that encode human annexin A2, thelight and heavy chains of an anti-annexin A2 antibody, and human factorH. Such polynucleotides may be used to deliver and express thecomplement stimulatory and inhibitory compositions described herein. Forexample, in certain embodiments, there are provided polynucleotidesencoding a fusion protein comprising an anti-annexin A2 antibody orantigen-binding fragment thereof and factor H or a biologically-activefragment thereof. In certain embodiments, the polynucleotides furthercomprise a sequence encoding a signal peptide operably linked at the 5′end of the sequence encoding the fusion protein. Exemplary nucleotidesequences of signal peptides are provided in SEQ ID NOs:12, 14, and 16.

Also provided herein are expression vectors comprising polynucleotidesexpressing any of the compositions described herein. The expressionvector can be used to direct expression of any of the compositionsdescribed herein in vitro or in vivo. The vector may include any elementnecessary to establish a conventional function of a vector, for example,a transcription promoter or terminator, a selectable marker, and anorigin of replication. The promoter can be constitutive or regulative,and is selected from, for example, promoters of genes for galactokinase(GAL1), uridylyltransferase (GAL7), epimerase (GAL 10), phosphoglyceratekinase (PGK), glyceraldehydes-3-phosphate dehydrogenase (GPD), alcoholdehydrogenase (ADH), and the like.

Many expression vectors are known to those of skill in the art. Forexample, E. coli may be transformed using pBR322, a plasmid derived froman E. coli species (Mandel et al., J. Mol. Biol., 53:154 (1970)).Plasmid pBR322 contains genes for ampicillin and tetracyclineresistance, and thus provides easy means for selection. Other vectorsinclude different features such as different promoters, which are oftenimportant in expression. For example, plasmids pKK223-3 (Pharmacia FineChemicals, Uppsala, Sweden), pKK233-2 (Clontech, Palo Alto, Calif.,USA), and pGEM1 (Promega Biotech, Madison, Wis., USA), are allcommercially available. Other vectors that are useful as describedherein include, but are not limited to, pET21a (Studier et al., MethodsEnzymol. (1990) 185: 60-89), pR1T5, and pR1T2T (PharmaciaBiotechnology), and pB0475 (Cunningham et al., Science, 243: 1330-1336(1989); U.S. Pat. No. 5,580,723). Mammalian expression vectors maycontain non-transcribed elements such as an origin of replication,promoter and enhancer, and 5′ or 3′ nontranslated sequences such asribosome binding sites, a polyadenylation site, acceptor site and splicedonor, and transcriptional termination sequences. Promoters for use inmammalian expression vectors usually are for example viral promoterssuch as Polyoma, Adenovirus, HTLV, Simian Virus 40 (SV 40), and humancytomegalovirus (CMV). Vectors can also be constructed using standardtechniques by combining the relevant traits of the vectors describedabove.

Also provided are host cells (such as isolated cells, transient celllines, and stable cell lines) for expressing any of the compositionsdescribed herein. The host cell may be prokaryotic or eukaryotic.Exemplary prokaryote host cells include E. coli K12 strain 294 (ATCC No.31446), E. coli B, E. coli X1776 (ATCC No. 31537), E. coli W3110 (F-,gamma-, prototrophic/ATCC No. 27325), Bacilli such as B. subtilis, andother Enterobacteriaceae such as Salmonella typhimurium or Serratiamarcesans and various Pseudomonas species. One suitable prokaryotic hostcell is E. coli BL21 (Stratagene Corp., La Jolla, Calif., USA), which isdeficient in the OmpT and Lon proteases, which may interfere withisolation of intact recombinant proteins, and useful with T7promoter-driven vectors, such as the pET vectors. Another suitableprokaryote is E. coli W3110 (ATCC No. 27325). When expressed byprokaryotes the peptides typically contain an N-terminal methionine or aformyl-methionine (“f-Met”) and are not glycosylated. In the case offusion proteins, the N-terminal methionine or f-Met resides on the aminoterminus of the expressed protein or of the signal sequence of theexpressed protein. These examples are, of course, intended to beillustrative rather than limiting.

In addition to prokaryotes, eukaryotic microbes such as filamentousfungi or yeast are suitable cloning or expression hosts forfusion-protein-encoding vectors. Saccharomyces cerevisiae is a commonlyused lower eukaryotic host microorganism. Others includeSchizosaccharomyces pombe (Beach and Nurse, Nature, 290: 140 (1981); EP139,383 published 2 May 1985); Kluyveromyces hosts (U.S. Pat. No.4,943,529; Fleer et al., Bio/Technology (1991) 9:968-975) such as, e.g.,K. lactis (MW98-8C, CBS683, CBS4574; Louvencourt et al., J. Bacteriol.,154(2):737-742 (1983)), K. fragilis (ATCC 12,424), K. bulgaricus (ATCCNo. 16,045), K. wickeramii (ATCC No. 24,178), K. waltii (ATCC No.56,500), K. drosophilarum (ATCC No. 36,906; Van den Berg et al.,Bio/Technology (1990) 8:135), K. thermotolerans, and K. marxianusyarrowia (EP 402,226); Pichia pastoris (EP 183,070; Sreekrishna et al.,J. Basic Microbiol. (1988) 28:265-278); Candida; Trichoderma reesia (EP244,234); Neurospora crassa (Case et al, Proc. Nat'l Acad. Sci. USA,(1979) 76:5259-5263); Schwanniomyces, such as Schwanniomycesoccidentalis (EP 394,538 published 31 Oct. 1990); and filamentous fungisuch as, e.g., Neurospora, Penicillium, Tolypocladium (WO 91/00357published 10 Jan. 1991), and Aspergillus hosts such as A. nidulans(Ballance et al., Biochem. Biophys. Res. Commun. (1983) 112:284-289;Tilburn et al., Gene (1983) 26:205-221; Yelton et al., Proc. Nat'l Acad.Sci. USA (1984) 81: 1470-1474) and A. niger (Kelly and Hynes, EMBO J.(1985) 4:475-479). Methylotropic yeasts are suitable herein and include,but are not limited to, yeast capable of growth on methanol selectedfrom the genera consisting of Hansenula, Candida, Kloeckera, Pichia,Saccharomyces, Torulopsis, and Rhodotorula. A list of specific speciesthat are exemplary of this class of yeasts may be found in C. Anthony,THE BIOCHEMISTRY OF METHYLOTROPHS, 269 (1982). Host cells also includeinsect cells such as Drosophila S2 and Spodoptera Sf9, as well as plantcells.

Examples of useful mammalian host cell lines include, but are notlimited to, HeLa, Chinese hamster ovary (CHO), COS-7, L cells, C127,3T3, BHK, CHL-1, NSO, HEK293, WI38, BHK, C127 or MDCK cell lines.Another exemplary mammalian cell line is CHL-1. When CHL-1 is used,hygromycin is included as a eukaryotic selection marker. CHL-1 cells arederived from RPMI 7032 melanoma cells, a readily available human cellline. Cells suitable for use as described herein are commerciallyavailable from the American Type Culture Collection (“ATCC”; Manassas,Va., USA).

In certain embodiments, the host cell is a non-human host cell. Incertain embodiments, the host cell is a CHO cell. In certainembodiments, the host cell is a 293 cell.

Any of the compositions described herein and prepared according torecombinant methods known in the art can be isolated by a variety ofmethods known in the art. In certain embodiments, when the compositionscomprise a secretory peptide operably linked to a polynucleotideencoding the desired composition and are thus secreted into the growthmedium, the molecule can be purified directly from the medium. If thefusion protein is not secreted, it can be isolated from cell lysates.Cell disruption can be done by any conventional method, includingfreeze-thaw cycling, sonication, mechanical disruption, or use of celllysing and/or chaotropic agents. The desired composition molecules canbe purified from cell lysates by various methods. These include, but arenot limited to, immunoaffinity chromatography, reverse phasechromatography, cation exchange chromatography, anion exchangechromatography, hydrophobic interaction chromatography, gel filtrationchromatography, and HPLC. For example, a fusion protein comprising ananti-annexin A2 antibody or antigen-binding fragment thereof and factorH or a biologically-active fragment thereof can be purified byimmunoaffinity chromatography using an antibody that recognizes thefactor H portion or an antibody that recognizes the Fc portion of theanti-annexin A2 antibody, or both. In certain embodiments, an antibodyrecognizing the first four N-terminal SCR domains of factor H is usedfor purifying the fusion protein. In certain embodiments, the desiredcomposition is purified by ion exchange chromatography.

The polypeptide may or may not be properly folded when expressed as afusion protein. These factors determine whether the fusion protein mustbe denatured and refolded, and if so, whether these procedures areemployed before or after cleavage. When denaturing and refolding areneeded, typically the peptide is treated with a chaotrope, such aguanidine HCl, and is then treated with a redox buffer, containing, forexample, reduced and oxidized dithiothreitol or glutathione at theappropriate ratios, pH, and temperature, such that the peptide isrefolded to its native structure.

Any of the compositions described herein may also contain a tag (such asa cleavable tag) for purification. This tag can be fused to theC-terminus or N-terminus of the composition, and can be used tofacilitate protein purification.

In certain embodiments, the compositions described herein could besynthesized de novo in whole or in part, using chemical methods wellknown in the art. For example, the component amino acid sequences can besynthesized by solid phase techniques, cleaved from the resin, andpurified by preparative high performance liquid chromatography followedby chemical linkage to form a desired polypeptide. The composition ofthe synthetic peptides may be confirmed by amino acid analysis orsequencing.

The purified compositions described herein can be assayed for theirdesired properties (i.e., the ability to bind annexin A2, factor H, orbreakdown products of complement protein C3, or the ability to inhibitactivity of the alternative complement pathway) using standard in vitroor in vivo assays. For example, binding of a biologically-active factorH fragment to a breakdown product of complement protein C3 can bedetermined by surface plasmon resonance. By way of example, kineticanalysis of the interaction of a biologically-active fragment of factorH comprising SCRs 1 to 4 with C3dg-biotin can be performed using surfaceplasmon resonance (“SPR”) measurements made on a BIAcore 3000 instrument(Biacore AB, Uppsala, Sweden). Human C3dg-biotin can be bound to thesurface of BIAcore streptavidin sensor chips by injecting C3dg-biotinover the surface of one flow cell of the chip. Binding can be evaluatedover a range of factor H fragment concentrations. Association of thefactor H fragment with the ligand can be monitored for a certain periodof time (such as 120 seconds), after which the complex is allowed todissociate in the presence of buffer only for an additional period oftime (such as 120 seconds). Binding of biologically-active factor Hfragments to C3dg-immobilized flow cells can be corrected for binding tocontrol flow cells. Binding data can be fitted to a 1:1 Langmuir bindingmodel using BIAevaluation Version 3.1 software (BIAcore) and evaluatedfor best fit. The kinetic dissociation profiles obtained can be used tocalculate on and off rates (k_(a) and k_(d)) and affinity constants(K_(D)) using the BIAevaluation Version 3.1 program. Other assay methodsfor ligand binding are known in the art and can also be used.

An in vitro zymosan complement assay can be used to determine complementinhibitory activity of the fusion protein compositions comprising ananti-annexin A2 antibody or antigen-binding fragment thereof fused tofactor H or a biologically-active fragment thereof, or to determinecomplement stimulatory activity of the biologically-active fragments offactor H described herein. Lysis of rabbit erythrocytes by serum inMg-EGTA is another measure of activity that can be used. Lysis inMg-EGTA of human or sheep erythrocytes that have had sialic acid removedprovides for additional measures of activity.

Methods of Identifying Factor H-Binding Proteins

As discussed above, inhibition of alternative complement pathwayactivity on cell surfaces by factor H requires that factor H properlybind to the cell surface. Thus, activation of the alternative pathway ona particular surface is strongly influenced by the affinity of factor Hfor that surface. Certain tissues or cell types require factor H toregulate alternative pathway activation on their surface. In some cases,different binding regions of the factor H protein may be necessary forcomplement regulation on those tissues or cell types. In others, thebinding of factor H to surfaces in particular tissues may be affected byas-yet-unidentified proteins. Identification of putative tissue-specificbinding partners of factor H may provide potential mechanisms formodulating, i.e., stimulating or inhibiting, activity of the alternativecomplement pathway in different tissues. Thus, in another aspect,methods of identifying factor H-binding proteins are provided.

In certain embodiments, the methods of identifying factor H-bindingproteins comprise (1) incubating full-length factor H or abiologically-active fragment thereof with a cell lysate prepared from atarget tissue, cell type, or cell line under conditions for a timesufficient to permit putative factor H ligands to bind full-lengthfactor H or a biologically-active fragment thereof; (2) purifying thecomplex comprising bound factor H or a biologically-active fragmentthereof and one or more factor H-binding proteins; (3) separating thefactor H-binding proteins by two dimensional polyacrylamide gelelectrophoresis; and (4) identifying factor H-binding proteins.Full-length factor H can be obtained by purification from plasma, or maybe prepared recombinantly, by expression in eukaryotic cells followed bypurification from culture medium or lysed cells.

The cell lysate can be prepared from any desired tissue that can beobtained from an individual, for example, renal, hepatic, pulmonary,bone marrow, lymph node, cardiac muscle, skeletal muscle, smooth muscle,epithelial, brain, or pancreatic tissue and the like. The cell lysatecan also be prepared from cell lines of any desired origin, for example,stem cells, including embryonic stem cells, renal, hepatic, pulmonary,cardiac muscle, skeletal muscle, smooth muscle, epithelial, brain, B orT lymphocyte, lymph nodes, or pancreatic cell lines and the like. Thoseskilled in the art can easily select an appropriate tissue or cell lineto use with the methods provided herein, based on the site ofalternative complement activation being studied.

In certain embodiments, the full-length factor H further comprises a tagto facilitate purification, both of full-length factor H orbiologically-active fragments thereof and of factor H orbiologically-active fragments thereof bound to one or more factor Hbinding proteins. Various tags commonly used to facilitate purificationare known to those skilled in the art. In certain embodiments, the tagto facilitate purification is a poly-histidine tag, a maltose-bindingprotein epitope, or biotin. In certain embodiments, the complexcomprising bound factor H is purified via chromatography over a matrixcomprising streptavidin, nickel-NTA-agarose, or an anti-maltose bindingprotein antibody. In certain embodiments, the complexes are purified viastreptavidin-labeled magnetic beads. In certain embodiments, the factorH binding proteins are identified by mass spectrometry. In certainembodiments, the mass spectrometry is tandem mass spectrometry(“MS-MS”). In certain embodiments, the mass spectrometry ismatrix-assisted laser desorption ionized time-of-flight (“MALDI-TOF”)mass spectrometry. In certain embodiments, labeled factor H is used inFar Western blot analysis to identify specific binding proteins that arefurther characterized by antibody-directed Western blot analysis orother methods known to those skilled in the art.

Pharmaceutical Compositions

Also provided herein are pharmaceutical compositions comprising annexinA2 or biologically-active fragments thereof, fusion proteins comprisinganti-annexin A2 antibodies or antigen-binding fragments thereof fused toDAF, factor H, MCP, CD59, CR1, mouse Crry protein or biologically-activefragments thereof, or factor H or biologically-active fragments thereofand a pharmaceutically acceptable carrier.

The pharmaceutical compositions may be suitable for a variety of modesof administration as described herein, including, for example, systemicor localized administration. The pharmaceutical compositions describedherein can be in the form of injectable solutions. The pharmaceuticalcompositions described herein can be packaged in single unit dosages orin multidosage forms.

In certain embodiments, the pharmaceutical compositions comprise annexinA2 or biologically-active fragments thereof and a pharmaceuticallyacceptable carrier suitable for administration to an individual. Incertain embodiments, the pharmaceutical compositions comprise fusionproteins comprising an anti-annexin A2 antibody or antigen-bindingfragment thereof fused to DAF, factor H, MCP, CD59, CR1, mouse Crryprotein or biologically-active fragments thereof and a pharmaceuticallyacceptable carrier suitable for administration to an individual.

In certain embodiments, the pharmaceutical composition comprises afusion protein comprising an anti-annexin A2 antibody or antigen-bindingfragment thereof fused to full-length human DAF (SEQ ID NO: 17) and apharmaceutically acceptable carrier suitable for administration to anindividual. In certain embodiments, the pharmaceutical compositioncomprises a fusion protein comprising an anti-annexin A2 antibody orantigen-binding fragment thereof fused to a biologically-active fragmentof human DAF comprising full-length human DAF lacking its GPI anchor(amino acids 35-353 of SEQ ID NO: 17) and a pharmaceutically acceptablecarrier suitable for administration to an individual. In certainembodiments, the pharmaceutical composition comprises a fusion proteincomprising an anti-annexin A2 antibody or antigen-binding fragmentthereof fused to a biologically-active fragment of human DAF comprisingSCRs 1-4 of human DAF (amino acids 35-285 of SEQ ID NO:17) and apharmaceutically acceptable carrier suitable for administration to anindividual.

In certain embodiments, the pharmaceutical composition comprises afusion protein comprising an anti-annexin A2 antibody or antigen-bindingfragment thereof fused to full-length factor H and a pharmaceuticallyacceptable carrier suitable for administration to an individual. Incertain embodiments, the pharmaceutical composition comprises a fusionprotein comprising an anti-annexin A2 antibody or antigen-bindingfragment thereof fused to a biologically-active fragment of factor Hcomprising SCRs 1 to 4 and a pharmaceutically acceptable carriersuitable for administration to an individual. In certain embodiments,the pharmaceutical composition comprises a fusion protein comprising ananti-annexin A2 antibody or antigen-binding fragment thereof fused to abiologically-active fragment of factor H comprising SCRs 1 to 8 and apharmaceutically acceptable carrier suitable for administration to anindividual. In certain embodiments, the pharmaceutical compositioncomprises a fusion protein comprising an anti-annexin A2 antibody orantigen-binding fragment thereof fused a biologically-active fragment offactor H comprising SCRs 1 to 18 and a pharmaceutically acceptablecarrier suitable for administration to an individual. In certainembodiments, the pharmaceutical compositions comprisebiologically-active fragments of factor H lacking the complementregulatory domain in SCRs 1 to 4 of full-length factor H and apharmaceutically acceptable carrier suitable for administration to anindividual. In certain embodiments, the biologically-active fragment offactor H comprises SCRs 19 to 20 of factor H and a pharmaceuticallyacceptable carrier suitable for administration to an individual.

In certain embodiments, the pharmaceutical composition comprises afusion protein comprising an anti-annexin A2 antibody or antigen-bindingfragment thereof fused to full-length human MCP (SEQ ID NO: 19) and apharmaceutically acceptable carrier suitable for administration to anindividual. In certain embodiments, the pharmaceutical compositioncomprises a fusion protein comprising an anti-annexin A2 antibody orantigen-binding fragment thereof fused to a biologically-active fragmentof human MCP comprising the extracellular domain of human MCP (aminoacids 35-343 of SEQ ID NO:19) and a pharmaceutically acceptable carriersuitable for administration to an individual. In certain embodiments,the pharmaceutical composition comprises a fusion protein comprising ananti-annexin A2 antibody or antigen-binding fragment thereof fused to abiologically-active fragment of human MCP comprising SCRs 1 to 4 ofhuman MCP (amino acids 35-285 of SEQ ID NO:19) and a pharmaceuticallyacceptable carrier suitable for administration to an individual.

In certain embodiments, the pharmaceutical composition comprises afusion protein comprising an anti-annexin A2 antibody or antigen-bindingfragment thereof fused to full-length human CD59 (SEQ ID NO:21) and apharmaceutically acceptable carrier suitable for administration to anindividual. In certain embodiments, the pharmaceutical compositioncomprises a fusion protein comprising an anti-annexin A2 antibody orantigen-binding fragment thereof fused to a biologically-active fragmentof human CD59 comprising the extracellular domain of human CD59 lackinga GPI anchor and/or the amino acid to which it is attached (i.e.,Asn-102)(amino acids 26-102 of SEQ ID NO:21) and a pharmaceuticallyacceptable carrier suitable for administration to an individual.

In certain embodiments, the pharmaceutical composition comprises afusion protein comprising an anti-annexin A2 antibody or antigen-bindingfragment thereof fused to full-length human CR1 (SEQ ID NO:23) and apharmaceutically acceptable carrier suitable for administration to anindividual. In certain embodiments, the pharmaceutical compositioncomprises a fusion protein comprising an anti-annexin A2 antibody orantigen-binding fragment thereof fused to a biologically-active fragmentof human CR1 comprising the complete extracellular domain of human CR1(SCRs 1 to 30)(amino acids 42-1971 of SEQ ID NO:23) and apharmaceutically acceptable carrier suitable for administration to anindividual. In certain embodiments, the pharmaceutical compositioncomprises a fusion protein comprising an anti-annexin A2 antibody orantigen-binding fragment thereof fused to a biologically active fragmentof full-length human CR1 comprising SCRs 1 to 4 (amino acids 42-295 ofSEQ ID NO:23) and a pharmaceutically acceptable carrier suitable foradministration to an individual. In certain embodiments, thepharmaceutical composition comprises a fusion protein comprising ananti-annexin A2 antibody or antigen-binding fragment thereof fused to abiologically active fragment of full-length human CR1 comprising SCRs 1to 11 (amino acids 42-745 of SEQ ID NO:23) and a pharmaceuticallyacceptable carrier suitable for administration to an individual. Incertain embodiments, the pharmaceutical composition comprises a fusionprotein comprising an anti-annexin A2 antibody or antigen-bindingfragment thereof fused to a biologically active fragment of full-lengthhuman CR1 comprising SCRs 1 to 18 (amino acids 42-1195 of SEQ ID NO:23)and a pharmaceutically acceptable carrier suitable for administration toan individual.

In certain embodiments, the pharmaceutical composition comprises afusion protein comprising an anti-annexin A2 antibody or antigen-bindingfragment thereof fused to full-length mouse Crry protein (SEQ ID NO:25)and a pharmaceutically acceptable carrier suitable for administration toan individual. In certain embodiments, the pharmaceutical compositioncomprises a fusion protein comprising an anti-annexin A2 antibody orantigen-binding fragment thereof fused to a biologically-active fragmentof full-length mouse Crry protein comprising the complete extracellulardomain of mouse Crry protein (amino acids 41-405 of SEQ ID NO:25) and apharmaceutically acceptable carrier suitable for administration to anindividual. In certain embodiments, the pharmaceutical compositioncomprises a fusion protein comprising an anti-annexin A2 antibody orantigen-binding fragment thereof fused to a biologically-active fragmentof full-length mouse Crry protein comprising SCRs 1 to 5 of mouse Crryprotein (amino acids 83-400 of SEQ ID NO:25) and a pharmaceuticallyacceptable carrier suitable for administration to an individual.

In certain embodiments, the pharmaceutical compositions comprise annexinA2 or biologically-active fragments thereof and a pharmaceuticallyacceptable carrier suitable for oral administration to an individual. Incertain embodiments, the pharmaceutical compositions comprise fusionproteins comprising an anti-annexin A2 antibody or antigen-bindingfragment thereof fused to DAF, factor H, MCP, CD59, CR1, mouse Crryprotein or biologically-active fragments thereof and a pharmaceuticallyacceptable carrier suitable for oral administration to an individual.

In certain embodiments, the pharmaceutical composition comprises afusion protein comprising an anti-annexin A2 antibody or antigen-bindingfragment thereof fused to full-length human DAF (SEQ ID NO: 17) and apharmaceutically acceptable carrier suitable for oral administration toan individual. In certain embodiments, the pharmaceutical compositioncomprises a fusion protein comprising an anti-annexin A2 antibody orantigen-binding fragment thereof fused to a biologically-active fragmentof human DAF comprising full-length human DAF lacking its GPI anchor(amino acids 35-353 of SEQ ID NO: 17) and a pharmaceutically acceptablecarrier suitable for oral administration to an individual. In certainembodiments, the pharmaceutical composition comprises a fusion proteincomprising an anti-annexin A2 antibody or antigen-binding fragmentthereof fused to a biologically-active fragment of human DAF comprisingSCRs 1-4 of human DAF (amino acids 35-285 of SEQ ID NO:17) and apharmaceutically acceptable carrier suitable for oral administration toan individual.

In certain embodiments, the pharmaceutical composition comprises afusion protein comprising an anti-annexin A2 antibody or antigen-bindingfragment thereof fused to full-length factor H and a pharmaceuticallyacceptable carrier suitable for oral administration to an individual. Incertain embodiments, the pharmaceutical composition comprises a fusionprotein comprising an anti-annexin A2 antibody or antigen-bindingfragment thereof fused to a biologically-active fragment of factor Hcomprising SCRs 1 to 4 and a pharmaceutically acceptable carriersuitable for oral administration to an individual. In certainembodiments, the pharmaceutical composition comprises a fusion proteincomprising an anti-annexin A2 antibody or antigen-binding fragmentthereof fused to a biologically-active fragment of factor H comprisingSCRs 1 to 8 and a pharmaceutically acceptable carrier suitable for oraladministration to an individual. In certain embodiments, thepharmaceutical composition comprises a fusion protein comprising ananti-annexin A2 antibody or antigen-binding fragment thereof fused abiologically-active fragment of factor H comprising SCRs 1 to 18 and apharmaceutically acceptable carrier suitable for oral administration toan individual. In certain embodiments, the pharmaceutical compositionscomprise biologically-active fragments of factor H lacking thecomplement regulatory domain in SCRs 1 to 4 of full-length factor H anda pharmaceutically acceptable carrier suitable for oral administrationto an individual. In certain embodiments, the biologically-activefragment of factor H comprises SCRs 19 to 20 of factor H and apharmaceutically acceptable carrier suitable for oral administration toan individual.

In certain embodiments, the pharmaceutical composition comprises afusion protein comprising an anti-annexin A2 antibody or antigen-bindingfragment thereof fused to full-length human MCP (SEQ ID NO: 19) and apharmaceutically acceptable carrier suitable for oral administration toan individual. In certain embodiments, the pharmaceutical compositioncomprises a fusion protein comprising an anti-annexin A2 antibody orantigen-binding fragment thereof fused to a biologically-active fragmentof human MCP comprising the extracellular domain of human MCP (aminoacids 35-343 of SEQ ID NO:19) and a pharmaceutically acceptable carriersuitable for oral administration to an individual. In certainembodiments, the pharmaceutical composition comprises a fusion proteincomprising an anti-annexin A2 antibody or antigen-binding fragmentthereof fused to a biologically-active fragment of human MCP comprisingSCRs 1 to 4 of human MCP (amino acids 35-285 of SEQ ID NO:19) and apharmaceutically acceptable carrier suitable for oral administration toan individual.

In certain embodiments, the pharmaceutical composition comprises afusion protein comprising an anti-annexin A2 antibody or antigen-bindingfragment thereof fused to full-length human CD59 (SEQ ID NO:21) and apharmaceutically acceptable carrier suitable for oral administration toan individual. In certain embodiments, the pharmaceutical compositioncomprises a fusion protein comprising an anti-annexin A2 antibody orantigen-binding fragment thereof fused to a biologically-active fragmentof human CD59 comprising the extracellular domain of human CD59 lackinga GPI anchor and/or the amino acid to which it is attached (i.e.,Asn-102)(amino acids 26-102 of SEQ ID NO:21) and a pharmaceuticallyacceptable carrier suitable for oral administration to an individual.

In certain embodiments, the pharmaceutical composition comprises afusion protein comprising an anti-annexin A2 antibody or antigen-bindingfragment thereof fused to full-length human CR1 (SEQ ID NO:23) and apharmaceutically acceptable carrier suitable for oral administration toan individual. In certain embodiments, the pharmaceutical compositioncomprises a fusion protein comprising an anti-annexin A2 antibody orantigen-binding fragment thereof fused to a biologically-active fragmentof human CR1 comprising the complete extracellular domain of human CR1(SCRs 1 to 30)(amino acids 42-1971 of SEQ ID NO:23) and apharmaceutically acceptable carrier suitable for oral administration toan individual. In certain embodiments, the pharmaceutical compositioncomprises a fusion protein comprising an anti-annexin A2 antibody orantigen-binding fragment thereof fused to a biologically active fragmentof full-length human CR1 comprising SCRs 1 to 4 (amino acids 42-295 ofSEQ ID NO:23) and a pharmaceutically acceptable carrier suitable fororal administration to an individual. In certain embodiments, thepharmaceutical composition comprises a fusion protein comprising ananti-annexin A2 antibody or antigen-binding fragment thereof fused to abiologically active fragment of full-length human CR1 comprising SCRs 1to 11 (amino acids 42-745 of SEQ ID NO:23) and a pharmaceuticallyacceptable carrier suitable for oral administration to an individual. Incertain embodiments, the pharmaceutical composition comprises a fusionprotein comprising an anti-annexin A2 antibody or antigen-bindingfragment thereof fused to a biologically active fragment of full-lengthhuman CR1 comprising SCRs 1 to 18 (amino acids 42-1195 of SEQ ID NO:23)and a pharmaceutically acceptable carrier suitable for oraladministration to an individual.

In certain embodiments, the pharmaceutical composition comprises afusion protein comprising an anti-annexin A2 antibody or antigen-bindingfragment thereof fused to full-length mouse Crry protein (SEQ ID NO:25)and a pharmaceutically acceptable carrier suitable for oraladministration to an individual. In certain embodiments, thepharmaceutical composition comprises a fusion protein comprising ananti-annexin A2 antibody or antigen-binding fragment thereof fused to abiologically-active fragment of full-length mouse Crry proteincomprising the complete extracellular domain of mouse Crry protein(amino acids 41-405 of SEQ ID NO:25) and a pharmaceutically acceptablecarrier suitable for oral administration to an individual. In certainembodiments, the pharmaceutical composition comprises a fusion proteincomprising an anti-annexin A2 antibody or antigen-binding fragmentthereof fused to a biologically-active fragment of full-length mouseCrry protein comprising SCRs 1 to 5 of mouse Crry protein (amino acids83-400 of SEQ ID NO:25) and a pharmaceutically acceptable carriersuitable for oral administration to an individual.

In certain embodiments, the pharmaceutical compositions comprise annexinA2 or biologically-active fragments thereof and a pharmaceuticallyacceptable carrier suitable for intravenous injection into anindividual. In certain embodiments, the pharmaceutical compositionscomprise fusion proteins comprising an anti-annexin A2 antibody orantigen-binding fragment thereof fused to DAF, factor H, MCP, CD59, CR1,mouse Crry protein or biologically-active fragments thereof and apharmaceutically acceptable carrier suitable for intravenous injectioninto an individual.

In certain embodiments, the pharmaceutical composition comprises afusion protein comprising an anti-annexin A2 antibody or antigen-bindingfragment thereof fused to full-length human DAF (SEQ ID NO:17) and apharmaceutically acceptable carrier suitable for intravenous injectioninto an individual. In certain embodiments, the pharmaceuticalcomposition comprises a fusion protein comprising an anti-annexin A2antibody or antigen-binding fragment thereof fused to abiologically-active fragment of human DAF comprising full-length humanDAF lacking its GPI anchor (amino acids 35-353 of SEQ ID NO: 17) and apharmaceutically acceptable carrier suitable for intravenous injectioninto an individual. In certain embodiments, the pharmaceuticalcomposition comprises a fusion protein comprising an anti-annexin A2antibody or antigen-binding fragment thereof fused to abiologically-active fragment of human DAF comprising SCRs 1-4 of humanDAF (amino acids 35-285 of SEQ ID NO:17) and a pharmaceuticallyacceptable carrier suitable for intravenous injection into anindividual.

In certain embodiments, the pharmaceutical composition comprises afusion protein comprising an anti-annexin A2 antibody or antigen-bindingfragment thereof fused to full-length factor H and a pharmaceuticallyacceptable carrier suitable for intravenous injection into anindividual. In certain embodiments, the pharmaceutical compositioncomprises a fusion protein comprising an anti-annexin A2 antibody orantigen-binding fragment thereof fused to a biologically-active fragmentof factor H comprising SCRs 1 to 4 and a pharmaceutically acceptablecarrier suitable for intravenous injection into an individual. Incertain embodiments, the pharmaceutical composition comprises a fusionprotein comprising an anti-annexin A2 antibody or antigen-bindingfragment thereof fused to a biologically-active fragment of factor Hcomprising SCRs 1 to 8 and a pharmaceutically acceptable carriersuitable for intravenous injection into an individual. In certainembodiments, the pharmaceutical composition comprises a fusion proteincomprising an anti-annexin A2 antibody or antigen-binding fragmentthereof fused a biologically-active fragment of factor H comprising SCRs1 to 18 and a pharmaceutically acceptable carrier suitable forintravenous injection into an individual. In certain embodiments, thepharmaceutical compositions comprise biologically-active fragments offactor H lacking the complement regulatory domain in SCRs 1 to 4 offull-length factor H and a pharmaceutically acceptable carrier suitablefor intravenous injection into an individual. In certain embodiments,the biologically-active fragment of factor H comprises SCRs 19 to 20 offactor H and a pharmaceutically acceptable carrier suitable forintravenous injection into an individual.

In certain embodiments, the pharmaceutical composition comprises afusion protein comprising an anti-annexin A2 antibody or antigen-bindingfragment thereof fused to full-length human MCP (SEQ ID NO: 19) and apharmaceutically acceptable carrier suitable for intravenous injectioninto an individual. In certain embodiments, the pharmaceuticalcomposition comprises a fusion protein comprising an anti-annexin A2antibody or antigen-binding fragment thereof fused to abiologically-active fragment of human MCP comprising the extracellulardomain of human MCP (amino acids 35-343 of SEQ ID NO: 19) and apharmaceutically acceptable carrier suitable for intravenous injectioninto an individual. In certain embodiments, the pharmaceuticalcomposition comprises a fusion protein comprising an anti-annexin A2antibody or antigen-binding fragment thereof fused to abiologically-active fragment of human MCP comprising SCRs 1 to 4 ofhuman MCP (amino acids 35-285 of SEQ ID NO:19) and a pharmaceuticallyacceptable carrier suitable for intravenous injection into anindividual.

In certain embodiments, the pharmaceutical composition comprises afusion protein comprising an anti-annexin A2 antibody or antigen-bindingfragment thereof fused to full-length human CD59 (SEQ ID NO:21) and apharmaceutically acceptable carrier suitable for intravenous injectioninto an individual. In certain embodiments, the pharmaceuticalcomposition comprises a fusion protein comprising an anti-annexin A2antibody or antigen-binding fragment thereof fused to abiologically-active fragment of human CD59 comprising the extracellulardomain of human CD59 lacking a GPI anchor and/or the amino acid to whichit is attached (i.e., Asn-102)(amino acids 26-102 of SEQ ID NO:21) and apharmaceutically acceptable carrier suitable for intravenous injectioninto an individual.

In certain embodiments, the pharmaceutical composition comprises afusion protein comprising an anti-annexin A2 antibody or antigen-bindingfragment thereof fused to full-length human CR1 (SEQ ID NO:23) and apharmaceutically acceptable carrier suitable for intravenous injectioninto an individual. In certain embodiments, the pharmaceuticalcomposition comprises a fusion protein comprising an anti-annexin A2antibody or antigen-binding fragment thereof fused to abiologically-active fragment of human CR1 comprising the completeextracellular domain of human CR1 (SCRs 1 to 30)(amino acids 42-1971 ofSEQ ID NO:23) and a pharmaceutically acceptable carrier suitable forintravenous injection into an individual. In certain embodiments, thepharmaceutical composition comprises a fusion protein comprising ananti-annexin A2 antibody or antigen-binding fragment thereof fused to abiologically active fragment of full-length human CR1 comprising SCRs 1to 4 (amino acids 42-295 of SEQ ID NO:23) and a pharmaceuticallyacceptable carrier suitable for intravenous injection into anindividual. In certain embodiments, the pharmaceutical compositioncomprises a fusion protein comprising an anti-annexin A2 antibody orantigen-binding fragment thereof fused to a biologically active fragmentof full-length human CR1 comprising SCRs 1 to 11 (amino acids 42-745 ofSEQ ID NO:23) and a pharmaceutically acceptable carrier suitable forintravenous injection into an individual. In certain embodiments, thepharmaceutical composition comprises a fusion protein comprising ananti-annexin A2 antibody or antigen-binding fragment thereof fused to abiologically active fragment of full-length human CR1 comprising SCRs 1to 18 (amino acids 42-1195 of SEQ ID NO:23) and a pharmaceuticallyacceptable carrier suitable for intravenous injection into anindividual.

In certain embodiments, the pharmaceutical composition comprises afusion protein comprising an anti-annexin A2 antibody or antigen-bindingfragment thereof fused to full-length mouse Crry protein (SEQ ID NO:25)and a pharmaceutically acceptable carrier suitable for intravenousinjection into an individual. In certain embodiments, the pharmaceuticalcomposition comprises a fusion protein comprising an anti-annexin A2antibody or antigen-binding fragment thereof fused to abiologically-active fragment of full-length mouse Crry proteincomprising the complete extracellular domain of mouse Crry protein(amino acids 41-405 of SEQ ID NO:25) and a pharmaceutically acceptablecarrier suitable for intravenous injection into an individual. Incertain embodiments, the pharmaceutical composition comprises a fusionprotein comprising an anti-annexin A2 antibody or antigen-bindingfragment thereof fused to a biologically-active fragment of full-lengthmouse Crry protein comprising SCRs 1 to 5 of mouse Crry protein (aminoacids 83-400 of SEQ ID NO:25) and a pharmaceutically acceptable carriersuitable for intravenous injection into an individual.

In certain embodiments, the pharmaceutical compositions comprise annexinA2 or biologically-active fragments thereof and a pharmaceuticallyacceptable carrier suitable for injection into the arteries (such as therenal arteries) of an individual. In certain embodiments, thepharmaceutical compositions comprise fusion proteins comprising ananti-annexin A2 antibody or antigen-binding fragment thereof fused toDAF, factor H, MCP, CD59, CR1, mouse Crry protein or biologically-activefragments thereof and a pharmaceutically acceptable carrier suitable forinjection into the arteries (such as the renal arteries) of anindividual.

In certain embodiments, the pharmaceutical composition comprises afusion protein comprising an anti-annexin A2 antibody or antigen-bindingfragment thereof fused to full-length human DAF (SEQ ID NO: 17) and apharmaceutically acceptable carrier suitable for injection into thearteries (such as the renal arteries) of an individual. In certainembodiments, the pharmaceutical composition comprises a fusion proteincomprising an anti-annexin A2 antibody or antigen-binding fragmentthereof fused to a biologically-active fragment of human DAF comprisingfull-length human DAF lacking its GPI anchor (amino acids 35-353 of SEQID NO:17) and a pharmaceutically acceptable carrier suitable forinjection into the arteries (such as the renal arteries) of anindividual. In certain embodiments, the pharmaceutical compositioncomprises a fusion protein comprising an anti-annexin A2 antibody orantigen-binding fragment thereof fused to a biologically-active fragmentof human DAF comprising SCRs 1-4 of human DAF (amino acids 35-285 of SEQID NO:17) and a pharmaceutically acceptable carrier suitable forinjection into the arteries (such as the renal arteries) of anindividual.

In certain embodiments, the pharmaceutical composition comprises afusion protein comprising an anti-annexin A2 antibody or antigen-bindingfragment thereof fused to full-length factor H and a pharmaceuticallyacceptable carrier suitable for injection into the arteries (such as therenal arteries) of an individual. In certain embodiments, thepharmaceutical composition comprises a fusion protein comprising ananti-annexin A2 antibody or antigen-binding fragment thereof fused to abiologically-active fragment of factor H comprising SCRs 1 to 4 and apharmaceutically acceptable carrier suitable for injection into thearteries (such as the renal arteries) of an individual. In certainembodiments, the pharmaceutical composition comprises a fusion proteincomprising an anti-annexin A2 antibody or antigen-binding fragmentthereof fused to a biologically-active fragment of factor H comprisingSCRs 1 to 8 and a pharmaceutically acceptable carrier suitable forinjection into the arteries (such as the renal arteries) of anindividual. In certain embodiments, the pharmaceutical compositioncomprises a fusion protein comprising an anti-annexin A2 antibody orantigen-binding fragment thereof fused a biologically-active fragment offactor H comprising SCRs 1 to 18 and a pharmaceutically acceptablecarrier suitable for injection into the arteries (such as the renalarteries) of an individual. In certain embodiments, the pharmaceuticalcompositions comprise biologically-active fragments of factor H lackingthe complement regulatory domain in SCRs 1 to 4 of full-length factor Hand a pharmaceutically acceptable carrier suitable for injection intothe arteries (such as the renal arteries) of an individual. In certainembodiments, the biologically-active fragment of factor H comprises SCRs19 to 20 of factor H and a pharmaceutically acceptable carrier suitablefor injection into the arteries (such as the renal arteries) of anindividual.

In certain embodiments, the pharmaceutical composition comprises afusion protein comprising an anti-annexin A2 antibody or antigen-bindingfragment thereof fused to full-length human MCP (SEQ ID NO: 19) and apharmaceutically acceptable carrier suitable for injection into thearteries (such as the renal arteries) of an individual. In certainembodiments, the pharmaceutical composition comprises a fusion proteincomprising an anti-annexin A2 antibody or antigen-binding fragmentthereof fused to a biologically-active fragment of human MCP comprisingthe extracellular domain of human MCP (amino acids 35-343 of SEQ IDNO:19) and a pharmaceutically acceptable carrier suitable for injectioninto the arteries (such as the renal arteries) of an individual. Incertain embodiments, the pharmaceutical composition comprises a fusionprotein comprising an anti-annexin A2 antibody or antigen-bindingfragment thereof fused to a biologically-active fragment of human MCPcomprising SCRs 1 to 4 of human MCP (amino acids 35-285 of SEQ ID NO:19) and a pharmaceutically acceptable carrier suitable for injectioninto the arteries (such as the renal arteries) of an individual.

In certain embodiments, the pharmaceutical composition comprises afusion protein comprising an anti-annexin A2 antibody or antigen-bindingfragment thereof fused to full-length human CD59 (SEQ ID NO:21) and apharmaceutically acceptable carrier suitable for injection into thearteries (such as the renal arteries) of an individual. In certainembodiments, the pharmaceutical composition comprises a fusion proteincomprising an anti-annexin A2 antibody or antigen-binding fragmentthereof fused to a biologically-active fragment of human CD59 comprisingthe extracellular domain of human CD59 lacking a GPI anchor and/or theamino acid to which it is attached (i.e., Asn-102)(amino acids 26-102 ofSEQ ID NO:21) and a pharmaceutically acceptable carrier suitable forinjection into the arteries (such as the renal arteries) of anindividual.

In certain embodiments, the pharmaceutical composition comprises afusion protein comprising an anti-annexin A2 antibody or antigen-bindingfragment thereof fused to full-length human CR1 (SEQ ID NO:23) and apharmaceutically acceptable carrier suitable for injection into thearteries (such as the renal arteries) of an individual. In certainembodiments, the pharmaceutical composition comprises a fusion proteincomprising an anti-annexin A2 antibody or antigen-binding fragmentthereof fused to a biologically-active fragment of human CR1 comprisingthe complete extracellular domain of human CR1 (SCRs 1 to 30)(aminoacids 42-1971 of SEQ ID NO:23) and a pharmaceutically acceptable carriersuitable for injection into the arteries (such as the renal arteries) ofan individual. In certain embodiments, the pharmaceutical compositioncomprises a fusion protein comprising an anti-annexin A2 antibody orantigen-binding fragment thereof fused to a biologically active fragmentof full-length human CR1 comprising SCRs 1 to 4 (amino acids 42-295 ofSEQ ID NO:23) and a pharmaceutically acceptable carrier suitable forinjection into the arteries (such as the renal arteries) of anindividual. In certain embodiments, the pharmaceutical compositioncomprises a fusion protein comprising an anti-annexin A2 antibody orantigen-binding fragment thereof fused to a biologically active fragmentof full-length human CR1 comprising SCRs 1 to 11 (amino acids 42-745 ofSEQ ID NO:23) and a pharmaceutically acceptable carrier suitable forinjection into the arteries (such as the renal arteries) of anindividual. In certain embodiments, the pharmaceutical compositioncomprises a fusion protein comprising an anti-annexin A2 antibody orantigen-binding fragment thereof fused to a biologically active fragmentof full-length human CR1 comprising SCRs 1 to 18 (amino acids 42-1195 ofSEQ ID NO:23) and a pharmaceutically acceptable carrier suitable forinjection into the arteries (such as the renal arteries) of anindividual.

In certain embodiments, the pharmaceutical composition comprises afusion protein comprising an anti-annexin A2 antibody or antigen-bindingfragment thereof fused to full-length mouse Crry protein (SEQ ID NO:25)and a pharmaceutically acceptable carrier suitable for injection intothe arteries (such as the renal arteries) of an individual. In certainembodiments, the pharmaceutical composition comprises a fusion proteincomprising an anti-annexin A2 antibody or antigen-binding fragmentthereof fused to a biologically-active fragment of full-length mouseCrry protein comprising the complete extracellular domain of mouse Crryprotein (amino acids 41-405 of SEQ ID NO:25) and a pharmaceuticallyacceptable carrier suitable for injection into the arteries (such as therenal arteries) of an individual. In certain embodiments, thepharmaceutical composition comprises a fusion protein comprising ananti-annexin A2 antibody or antigen-binding fragment thereof fused to abiologically-active fragment of full-length mouse Crry proteincomprising SCRs 1 to 5 of mouse Crry protein (amino acids 83-400 of SEQID NO:25) and a pharmaceutically acceptable carrier suitable forinjection into the arteries (such as the renal arteries) of anindividual. In certain embodiments, the pharmaceutically acceptablecarrier is suitable for oral administration to an individual.

For oral administration, the pharmaceutical compositions can take theform of, for example, tablets or capsules prepared by conventional meanswith pharmaceutically acceptable excipients such as binding agents(e.g., pregelatinized maize starch, polyvinylpyrrolidone orhydroxypropyl methylcellulose); fillers (e.g., lactose, microcrystallinecellulose or calcium hydrogen phosphate); lubricants (e.g., magnesiumstearate, talc or silica); disintegrants (e.g., potato starch or sodiumstarch glycolate); or wetting agents (e.g., sodium lauryl sulfate).Liquid preparations for oral administration can take the form of, forexample, solutions, syrups or suspensions, or they can be presented as adry product for constitution with water or other suitable vehicle beforeuse. Such liquid preparations can be prepared by conventional means withpharmaceutically acceptable additives such as suspending agents (e.g.,sorbitol syrup, cellulose derivatives or hydrogenated edible fats);emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles(e.g., ationd oil, oily esters, ethyl alcohol or fractionated vegetableoils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoates orsorbic acid). The preparations can also contain buffer salts, flavoring,coloring and sweetening agents as appropriate.

The liquid compositions are generally formulated as sterile,substantially isotonic solutions in full compliance with all GoodManufacturing Practice (GMP) regulations of the U.S. Food and DrugAdministration. In certain embodiments, the composition is free ofpathogen. For injection, the pharmaceutical composition can be in theform of liquid solutions, for example in physiologically compatiblebuffers such as Hank's Balanced Salt Solution, Phosphate-Buffered Salineor Ringer's solution. In addition, the pharmaceutical compositionsprovided herein can be in solid form and redissolved or resuspendedimmediately prior to use. Lyophilized compositions are alsocontemplated.

In certain embodiments, the composition is formulated in accordance withroutine procedures as a pharmaceutical composition adapted forinjection. In certain embodiments, the pharmaceutical compositionsprovided herein are formulated for intravenous, intraperitoneal, orintraocular injection. Typically, compositions for injection aresolutions in sterile isotonic aqueous buffer. Where necessary, thecomposition may also include a solubilizing agent and a local anestheticsuch as lignocaine to ease pain at the site of the injection. Generally,the ingredients are supplied either separately or mixed together in unitdosage form, for example, as a dry lyophilized powder or water-freeconcentrate in a hermetically sealed container such as an ampoule orsachette indicating the quantity of active agent. Where the compositionis to be administered by infusion, it can be dispensed with an infusionbottle containing sterile pharmaceutical grade water or saline. Wherethe composition is administered by injection, an ampoule of sterilewater for injection or saline can be provided so that the ingredientsmay be mixed prior to administration.

The pharmaceutical compositions may further comprise additionalingredients, for example preservatives, buffers, tonicity agents,antioxidants and stabilizers, nonionic wetting or clarifying agents,viscosity-increasing agents, and the like.

Suitable preservatives for use in a solution include polyquaternium-1,benzalkonium chloride, thimerosal, chlorobutanol, methyl paraben, propylparaben, phenylethyl alcohol, disodium-EDTA, sorbic acid, benzethoniumchloride, and the like. Typically (but not necessarily) suchpreservatives are employed at a level of from 0.001% to 1.0% by weight.

Suitable buffers include boric acid, sodium and potassium bicarbonate,sodium and potassium borates, sodium and potassium carbonate, sodiumacetate, sodium biphosphate and the like, in amounts sufficient tomaintain the pH at between about pH 6 and pH 8, and preferably, betweenabout pH 7 and pH 7.5.

Suitable tonicity agents include dextran 40, dextran 70, dextrose,glycerin, potassium chloride, propylene glycol, sodium chloride, and thelike, such that the sodium chloride equivalent of the injectablesolution is in the range 0.9 plus or minus 0.2%.

Suitable antioxidants and stabilizers include sodium bisulfite, sodiummetabisulfite, sodium thiosulfite, thiourea and the like. Suitablewetting and clarifying agents include polysorbate 80, polysorbate 20,poloxamer 282 and tyloxapol. Suitable viscosity-increasing agentsinclude dextran 40, dextran 70, gelatin, glycerin,hydroxyethylcellulose, hydroxmethylpropylcellulose, lanolin,methylcellulose, petrolatum, polyethylene glycol, polyvinyl alcohol,polyvinylpyrrolidone, carboxymethylcellulose and the like.

The pharmaceutical compositions may be suitable for a variety of modesof administration described herein, including for example systemic orlocalized administration. The pharmaceutical compositions can be in theform of eye drops, injectable solutions, or in a form suitable forinhalation (either through the mouth or the nose) or oraladministration. The pharmaceutical compositions described herein can bepackaged in single unit dosages or in multidosage forms.

In certain embodiments, the pharmaceutical compositions comprise annexinA2 or biologically-active fragments thereof and a pharmaceuticallyacceptable carrier suitable for administration to a human. In certainembodiments, the pharmaceutical compositions comprise fusion proteinscomprising an anti-annexin A2 antibody or antigen-binding fragmentthereof fused to factor H or a biologically-active fragment thereof anda pharmaceutically acceptable carrier suitable for administration to ahuman. In certain embodiments, the pharmaceutical composition comprisesa fusion protein comprising an anti-annexin A2 antibody orantigen-binding fragment thereof fused to full-length factor H and apharmaceutically acceptable carrier suitable for administration to ahuman. In certain embodiments, the pharmaceutical composition comprisesa fusion protein comprising an anti-annexin A2 antibody orantigen-binding fragment thereof fused to a biologically-active fragmentof factor H comprising SCRs 1 to 4 and a pharmaceutically acceptablecarrier suitable for administration to a human. In certain embodiments,the pharmaceutical composition comprises a fusion protein comprising ananti-annexin A2 antibody or antigen-binding fragment thereof fused to abiologically-active fragment of factor H comprising SCRs 1 to 8 and apharmaceutically acceptable carrier suitable for administration to ahuman. In certain embodiments, the pharmaceutical composition comprisesa fusion protein comprising an anti-annexin A2 antibody orantigen-binding fragment thereof fused to a biologically-active fragmentof factor H comprising SCRs 1 to 18 and a pharmaceutically acceptablecarrier suitable for administration to a human.

In certain embodiments, the pharmaceutical compositions comprisebiologically-active fragments of factor H lacking the complementregulatory domain in SCRs 1 to 4 of full-length factor H and apharmaceutically acceptable carrier suitable for administration to ahuman. In certain embodiments, the biologically-active fragment offactor H comprises SCRs 19 to 20 of factor H and a pharmaceuticallyacceptable carrier suitable for administration to a human. In certainembodiments, the pharmaceutical compositions comprisingbiologically-active fragments of factor H lacking the complementregulatory domain in SCRs 1 to 4 of full-length factor H and apharmaceutically acceptable carrier are suitable for administration toan individual or a human by any route of administration describedherein, including oral administration, intravenous injection, orinjection into the arteries, such as the renal arteries. In certainembodiments, the pharmaceutical compositions comprising abiologically-active fragment of factor H comprising SCRs 19 to 20 offactor H and a pharmaceutically acceptable carrier are suitable foradministration to an individual or a human by any route ofadministration described herein, including oral administration,intravenous injection, or injection into the arteries, such as the renalarteries.

In certain embodiments, the pharmaceutical compositions comprise annexinA2 or biologically-active fragments thereof and a pharmaceuticallyacceptable carrier suitable for intraocular injection in an individual.In certain embodiments, the pharmaceutical compositions comprise fusionproteins comprising an anti-annexin A2 antibody or antigen-bindingfragment thereof fused to factor H or a biologically-active fragmentthereof and a pharmaceutically acceptable carrier suitable forintraocular injection in an individual. In certain embodiments, thepharmaceutical composition comprises a fusion protein comprising ananti-annexin A2 antibody or antigen-binding fragment thereof fused tofull-length factor H and a pharmaceutically acceptable carrier suitablefor intraocular injection in an individual. In certain embodiments, thepharmaceutical composition comprises a fusion protein comprising ananti-annexin A2 antibody or antigen-binding fragment thereof fused to abiologically-active fragment of factor H comprising SCRs 1 to 4 and apharmaceutically acceptable carrier suitable for intraocular injectionin an individual. In certain embodiments, the pharmaceutical compositioncomprises a fusion protein comprising an anti-annexin A2 antibody orantigen-binding fragment thereof fused to a biologically-active fragmentof factor H comprising SCRs 1 to 8 and a pharmaceutically acceptablecarrier suitable for intraocular injection in an individual. In certainembodiments, the pharmaceutical composition comprises a fusion proteincomprising an anti-annexin A2 antibody or antigen-binding fragmentthereof fused a biologically-active fragment of factor H comprising SCRs1 to 18 and a pharmaceutically acceptable carrier suitable forintraocular injection in an individual. In certain embodiments, thepharmaceutical compositions comprise biologically-active fragments offactor H lacking the complement regulatory domain in SCRs 1 to 4 offull-length factor H and a pharmaceutically acceptable carrier suitablefor intraocular injection in an individual. In certain embodiments, thebiologically-active fragment of factor H comprises SCRs 19 to 20 offactor H and a pharmaceutically acceptable carrier suitable forintraocular injection in an individual.

In certain embodiments, the pharmaceutical compositions comprise annexinA2 or biologically-active fragments thereof and a pharmaceuticallyacceptable carrier suitable for topical administration to the eye of anindividual. In certain embodiments, the pharmaceutical compositionscomprise fusion proteins comprising an anti-annexin A2 antibody orantigen-binding fragment thereof fused to factor H or abiologically-active fragment thereof and a pharmaceutically acceptablecarrier suitable for topical administration to the eye of an individual.In certain embodiments, the pharmaceutical composition comprises afusion protein comprising an anti-annexin A2 antibody or antigen-bindingfragment thereof fused to full-length factor H and a pharmaceuticallyacceptable carrier suitable for topical administration to the eye of anindividual. In certain embodiments, the pharmaceutical compositioncomprises a fusion protein comprising an anti-annexin A2 antibody orantigen-binding fragment thereof fused to a biologically-active fragmentof factor H comprising SCRs 1 to 4 and a pharmaceutically acceptablecarrier suitable for topical administration to the eye of an individual.In certain embodiments, the pharmaceutical composition comprises afusion protein comprising an anti-annexin A2 antibody or antigen-bindingfragment thereof fused to a biologically-active fragment of factor Hcomprising SCRs 1 to 8 and a pharmaceutically acceptable carriersuitable for topical administration to the eye of an individual. Incertain embodiments, the pharmaceutical composition comprises a fusionprotein comprising an anti-annexin A2 antibody or antigen-bindingfragment thereof fused a biologically-active fragment of factor Hcomprising SCRs 1 to 18 and a pharmaceutically acceptable carriersuitable for topical administration to the eye of an individual. Incertain embodiments, the pharmaceutical compositions comprisebiologically-active fragments of factor H lacking the complementregulatory domain in SCRs 1 to 4 of full-length factor H and apharmaceutically acceptable carrier suitable for topical administrationto the eye of an individual. In certain embodiments, thebiologically-active fragment of factor H comprises SCRs 19 to 20 offactor H and a pharmaceutically acceptable carrier suitable for topicaladministration to the eye of an individual.

There are provided herein in certain embodiments compositions comprisingannexin A2 or biologically-active fragments thereof, fusion proteinscomprising an anti-annexin A2 antibody or antigen-binding fragmentthereof fused to factor H or a biologically-active fragment thereof, orbiologically-active fragments of factor H and a pharmaceuticallyacceptable carrier suitable for administration to the eye. Suchpharmaceutical carriers can be sterile liquids, such as water and oil,including those of petroleum, animal, vegetable or synthetic origin,such as peanut oil, soybean oil, mineral oil, and the like. Salinesolutions and aqueous dextrose, polyethylene glycol (PEG) and glycerolsolutions can also be employed as liquid carriers, particularly forinjectable solutions. Suitable pharmaceutical excipients include starch,glucose, lactose, sucrose, gelatin, malt, rice, sodium state, glycerolmonostearate, glycerol, propylene, water, and the like. Thepharmaceutical composition, if desired, can also contain minor amountsof wetting or emulsifying agents, or pH buffering agents. The annexin A2or biologically-active fragments thereof, fusion proteins comprising ananti-annexin A2 antibody or antigen-binding fragment thereof fused tofactor H or a biologically-active fragment thereof, orbiologically-active fragments of factor H and other components of thecomposition may be encased in polymers or fibrin glues to providecontrolled release of the molecule. These compositions can take the formof solutions, suspensions, emulsions, ointment, gel, or other solid orsemisolid compositions, and the like. The compositions typically have apH in the range of 4.5 to 8.0. The compositions must also be formulatedto have osmotic values that are compatible with the aqueous humor of theeye and ophthalmic tissues. Such osmotic values will generally be in therange of from about 200 to about 400 milliosmoles per kilogram of water(mOsm/kg), but will preferably be about 300 mOsm/kg.

The use of viscosity enhancing agents to provide topical compositionswith viscosities greater than the viscosity of simple aqueous solutionsmay be desirable to increase ocular absorption of the active compoundsby the target tissues or increase the retention time in the eye. Thus,in certain embodiments, the compositions further comprise viscosityenhancing agents. Such viscosity enhancing agents include, for example,polyvinyl alcohol, polyvinyl pyrrolidone, methyl cellulose, hydroxypropyl methylcellulose, hydroxyethyl cellulose, carboxymethyl cellulose,hydroxy propyl cellulose or other agents know to those skilled in theart. Such agents are typically employed at a level of from 0.01% to 2%by weight.

Methods of Modulating Activity of the Alternative Complement Pathway

In another aspect, there are provided methods of modulating, i.e.,stimulating or inhibiting, alternative complement activity in anindividual. In certain embodiments, the methods comprise methods ofinhibiting alternative complement activity. In certain embodiments, thealternative complement activity is associated with inflammation. Incertain embodiments, the inflammation is renal inflammation. In certainembodiments, the renal inflammation is associated withmembranoproliferative glomerulonephritis type II (MPGN II), hemolyticuremic syndrome, atypical hemolytic uremic syndrome, thromboticthrombocytopenic purpura, ischemia reperfusion (I/R) injury, andischemic acute kidney injury. In certain embodiments, the methodscomprise methods of inhibiting alternative complement activityassociated with drusen-related disease. In certain embodiments, thedrusen-related diseases are selected from the group consisting ofmembranoproliferative glomerulonephritis type II (MPGN II), age-relatedmacular degeneration, and amyloidosis.

In certain embodiments, the methods comprise methods of stimulatingalternative complement activity. In certain embodiments, the methodsstimulate alternative complement by administering a compositioncomprising a biologically-active fragment of factor H comprising SCRs 19and 20 in conjunction with a monoclonal antibody-based therapeutic. Incertain embodiments, the monoclonal antibody-based therapeutic is ananti-cancer agent. In other embodiments, the methods stimulatealternative complement by administering a composition comprising anannexin A2 or a biologically-active fragment thereof.

As used herein, the term “ischemia reperfusion (I/R) injury” refers toinflammatory injury to the endothelium and underlying parenchymaltissues following reperfusion of hypoxic tissues. It is a generalsyndrome that is responsible for both acute and chronic injury tovarious tissues including, for example, myocardium, central nervoussystem, hind limb and intestine. Ischemia reperfusion injury can resultin necrosis and irreversible cell injury. The complement pathway(including the alternative complement pathway) is a major mediator ofI/R injury. The methods provided herein are thus useful for inhibitingalternative complement-mediated inflammation associated with ischemiareperfusion, particularly that occurring in renal ischemia-reperfusioninjury. Ischemia-reperfusion injury can also occur in conjunction with avariety of other conditions including, but not limited to, stroke,spinal cord injury, trauma-induced hypovolemic shock, and autoimmunediseases such as rheumatoid arthritis (e.g., which can be greatlyworsened by ischemic injury of the synovium) or a variety of otherinflammatory diseases (diseases mediated by inflammation or whereininflammation is a symptom that may result in or be associated withischemic events and reperfusion). Other conditions and diseases in whichischemia-reperfusion injury occurs will be known to those of skill inthe art.

As used herein, the term “ischemic acute kidney injury” or “AKI” refersto an I/R-associated injury in rodents (J. M. Thurman et al., J.Immunol. (2003) 170:1517-1523; J. M. Thurman et al., J. Am. Soc.Nephrol. (2006) 17:707-715) and in humans (J. M. Thurman et al., KidneyInt. (2005) 67:524-530) that is associated with activation of thealternative pathway on the basolateral surface of injured tubular cells.Complement receptor 1-related gene/protein y (Crry, a rodent analog ofhuman MCP and CR1) is the only CRP expressed by proximal tubularepithelial cells in mice. I/R causes reduced surface expression of thisprotein. See J. M. Thurman et al., J. Clin. Invest. (2006) 116:357-368.Mice with congenital deficiency of Crry (Crry+/−) are more sensitivethan wild-type controls to ischemic acute renal failure (Id.),highlighting the importance of basolateral Crry for controlling thealternative pathway on this surface. Uncontrolled activation of thealternative pathway in the setting of reduced surface Crry indicatesthat circulating factor H has a limited ability to protect the surfaceof hypoxic tubular epithelial cells in rodents and humans.

As used herein, the term “hemolytic uremic syndrome” or “HUS” refers toa disease characterized by microangiopathic hemolytic anemia andthrombocytopenia, ultimately resulting in acute renal failure, caused bycontinuous platelet degradation in the periphery and platelet thrombinin the microcirculation of the kidney. See Zipfel, Seminars inThrombosis Hemostasis (2001) 27(3): 191-199. There is now considerableevidence that the nondiarrheal form of HUS (also known as atypical HUS,or aHUS) is associated with alternations and mutations of FH. Inaddition, autoantibodies to FH have been reported in aHUS patients.Thus, evidence suggests that the alternative complement pathway isinvolved in the development and progression of HUS and aHUS.

As used herein, the term “thrombotic thrombocytopenic purpura” or “TTP”refers to a rare disease characterized by microangiopathic hemolyticanemia, causing blood clots to form in small blood vessels throughoutthe body. These blood clots can cause serious problems if they blockblood vessels and limit blood flow to the brain, kidneys, or heart.Blood clots form when blood cells called platelets clump together.Formation of the blood clots characteristic of TTP depletes circulatingplatelet levels, sometimes resulting in bleeding into the skin(purpura), prolonged bleeding from cuts, and internal bleeding. It alsocauses small blood clots to form suddenly throughout the body, includingin the brain and kidneys.

Annexin A2 has also been identified as a component of drusen in monkeysaffected with both early- and late-onset macular degeneration,suggesting that the methods provided herein may also be used to inhibitalternative complement activity in drusen-associated diseases. See S.Umeda et al., FASEB J. (2005) 19(12):1683-1685. As used herein, the term“drusen-associated disease” refers to any disease in which formation ofdrusen or drusen-like extracellular disease plaque takes place, and forwhich drusen or drusen-like extracellular disease plaque causes orcontributes to thereto or represents a sign thereof. For example,age-related macular degeneration (AMD), characterized by the formationof macular drusen, is considered a drusen-associated disease. Non-oculardrusen-related disease include, but are not limited to, amyloidosis anddense deposit disease (i.e., type II membranoproliferativeglomerulonephritis).

The methods provided herein may also be used to treat alternativecomplement-mediated inflammation in drusen-associated diseases. As usedherein, the term “drusen-associated disease” refers to any disease inwhich formation of drusen or drusen-like extracellular disease plaquetakes place, and for which drusen or drusen-like extracellular diseaseplaque causes or contributes to thereto or represents a sign thereof.For example, age-related macular degeneration (AMD), characterized bythe formation of macular drusen, is considered a drusen-associateddisease. Non-ocular drusen-related disease include, but are not limitedto, amyloidosis, elastosis, dense deposit disease, and/oratherosclerosis. The term “drusen-related disease” also includesglomerulonephritis, such as MPGN II.

As used herein, the term “membranoproliferative glomerulonephritis typeII” or “MPGN II”, or “dense deposit disease” refers to a rare kidneydisease leading to persistent proteinuria, hematuria, and nephriticsyndrome. Factor H deficiency and dysfunction in MPGN II have beenreported in several cases. For example, mutations in factor H have beenfound in human patients with MPGN II. Pigs of the Norwegian Yorkshirebreed have factor H defects that are inherited in a recessive pattern.These animals develop MPGN II, show massive complement deposits in therenal glomeruli and die at an early age because of the renal failure. Insome cases, those complement deposits resemble the drusen characteristicof age-related macular degeneration. As discussed above, annexin A2 hasbeen shown to be a component of drusen. Furthermore, an autoantibodythat recognizes factor H has been described in a patient withhypocomplementemic MPGN II. Thus, evidence suggests that the alternativecomplement pathway is involved in the development and progression ofMPGN II.

As used herein, the term “age-related macular degeneration” or “AMD”refers to a disorder clinically characterized by progressive loss ofcentral vision which occurs as a result of damage to the photoreceptorcells in an area of the retina called the macula. AMD has been broadlyclassified into two clinical states: a wet form and a dry form, with thedry form making up to 80-90% of total cases. The dry form ischaracterized clinically by the presence of macular drusen, which arelocalized deposits between the retinal pigment epithelium (RPE) and theBruch's membrane, and by geographic atrophy characterized by RPE celldeath with overlying photoreceptor atrophy. Wet AMD, which accounts forapproximately 90% of serious vision loss, is associated withneovascularization in the area of the macular and leakage of these newvessels. The accumulation of blood and fluid can cause retina detachmentfollowed by rapid photoreceptor degeneration and loss of vision. It isgenerally accepted that the wet form of AMD is preceded by and arisesfrom the dry form. Analysis of the contents of drusen in AMD patientshas identified a large number of inflammatory proteins including amyloidproteins, annexin A2, coagulation factors, and a large number ofproteins of the complement pathway. A genetic variation in thecomplement factor H substantially raises the risk of age-related maculardegeneration (AMD), suggesting that uncontrolled complement activationunderlies the pathogenesis of AMD. See Edward et al., Science (2005)308:421; Haines et al., Science (2005) 308:419; Klein et al., Science308:385-389; Hageman et al., Proc. Natl. Acad. Sci. USA (2005),102:7227.

As used herein, the term “amyloidosis” refers to a group of conditionsin which abnormal organ or tissue deposits of amyloid proteins causedisease. Various proteins take on “amyloid form” due to characteristicalterations in their secondary structure. The term “amyloidosis” refersto a histological finding of amyloid deposits occurring in a number ofdifferent disease processes. Amyloidoses can be systemic (affecting manydifferent organ systems) or organ-specific. Some are inherited as aresult of mutations in the amyloid precursor protein, while othersresult from different diseases causing overabundant or abnormal proteinproduction-such as with over production of immunoglobulin light chainsin multiple myeloma (referred to as “AL amyloid”).

In certain embodiments, the methods comprise methods of inhibitingalternative complement activity. In certain embodiments, the alternativecomplement activity is associated with inflammation. Complement-mediatedinflammation associated with many diseases in which any of the threecomplement pathways is implicated can be treated by the methods asdescribed herein. Such diseases include, for example: (1) tissue damagedue to ischemia-reperfusion following acute myocardial infarction,aneurysm, stroke, hemorrhagic shock, crush injury, multiple organfailure, hypovolemic shock intestinal ischemia, spinal cord injury, andtraumatic brain injury; (2) inflammatory disorders, e.g., burns,endotoxemia and septic shock, adult respiratory distress syndrome,cardiopulmonary bypass, hemodialysis; anaphylactic shock, severe asthma,angioedema, Crohn's disease, sickle cell anemia, poststreptococcalglomerulonephritis, membraneous nephritis, and pancreatitis; (3)transplant rejection, e.g., hyperacute xenograft rejection; (4)pregnancy related diseases such as recurrent fetal loss andpre-eclampsia, and (5) adverse drug reactions, e.g., drug allergy, IL-2induced vascular leakage syndrome and radiographic contrast mediaallergy. Alternative complement-mediated inflammation associated withautoimmune disorders including, but not limited to, myasthenia gravis,Alzheimer's disease, multiple sclerosis, rheumatoid arthritis, systemiclupus erythematosus, insulin-dependent diabetes mellitus, acutedisseminated encephalomyelitis, Addison's disease, antiphospholipidantibody syndrome, autoimmune hepatitis, Crohn's disease, Goodpasture'ssyndrome, Graves' disease, Guillain-Barré syndrome, Hashimoto's disease,idiopathic thrombocytopenic purpura, pemphigus, Sjögren's syndrome, andTakayasu's arteritis, may also be treated with the compositionsdescribed herein.

In certain embodiments, the alternative complement-mediated inflammationto be treated by the methods provided herein is associated with any ofthe following disorders: post cardiopulmonary bypass complications;myocardial infarction; ischemia/reperfusion injury; stroke; acuterespiratory distress syndrome (ARDS); sepsis; burn injury; inflammationassociated with cardiopulmonary bypass and hemodialysis; plasmapheresis;plateletpheresis; leukopheresis; extracorporeal membrane oxygenation(ECMO); heparin-induced extracorporeal LDL precipitation (HELP);radiographic contrast media-induced allergic response; transplantrejection; and other inflammatory conditions and autoimmune/immunecomplex diseases such as multiple sclerosis, myasthemia gravis,pancreatitis, rheumatoid arthritis, Alzheimer's disease, asthma, thermalinjury, anaphylactic shock, bowel inflammation, urticaria, angioedema,vasculitis, glomerulonephritis, and Sjögren's syndrome, systemic lupuserythromatosus and lupus nephritis.

Rheumatoid arthritis is a chronic disease which can exhibit a variety ofsystemic manifestations. This disease has an unknown etiology andcharacteristically exhibits a persistent inflammatory synovitis whichusually involves peripheral joints in a symmetric distribution. The mostimportant feature of this incurable condition is complement-mediatedinflammation which causes cartilage destruction, bone erosions and,ultimately, joint deformities that are the hallmark of the disease.

In certain embodiments, there is provided a method of modulatingalternative complement activity in an individual, comprisingadministering to the individual a composition selected from the groupconsisting of (a) annexin A2 or a biologically-active fragment thereof;(b) a fusion protein comprising an anti-annexin A2 antibody or anantigen-binding fragment thereof fused to DAF, factor H, MCP, CD59, CR1,or mouse Crry protein or a biologically-active fragment thereof; and (c)a biologically-active fragment of factor H lacking the complementregulatory domain in SCRs 1 to 4 of full-length factor H. In certainembodiments, the individual is a mammal. In certain embodiments, themammal is a human, a mouse, or a rat. In certain embodiments,alternative complement activity is inhibited in an individual. Incertain embodiments, the individual is administered a compositionselected from the group consisting of (a) annexin A2 or abiologically-active fragment thereof; and (b) a fusion proteincomprising an anti-annexin A2 antibody or antigen-binding fragmentthereof fused to DAF, factor H, MCP, CD59, CR1, mouse Crry protein or abiologically-active fragment thereof. In certain embodiments, theindividual is administered a composition comprising annexin A2 or abiologically-active fragment thereof. In certain embodiments, theindividual is administered a composition comprising annexin A2. Incertain embodiments, the composition is administered orally or byinjection. In certain embodiments, the composition is injectedintravenously.

In certain embodiments, alternative complement activity is inhibited inan individual, and the individual is administered a compositioncomprising a fusion protein comprising an anti-annexin A2 antibody orantigen-binding fragment thereof fused to DAF, factor H, MCP, CD59, CR1,mouse Crry protein or a biologically-active fragment thereof. In certainembodiments, the antigen-binding fragment thereof comprises an Fab,Fab′, or F(ab′)₂ fragment. In certain embodiments, the biologicallyactive fragment of DAF fused to an anti-annexin A2 antibody orantigen-binding fragment thereof comprises the mature human DAF protein(amino acids 35-353 of SEQ ID NO:17) without its GPI anchor. In certainembodiments, the biologically active fragment of human DAF fused to ananti-annexin A2 antibody or antigen-binding fragment thereof comprisesshort consensus repeat sequences 1 to 4 (SCRs 1 to 4) of full-lengthhuman DAF (amino acids 35 to 285 of SEQ ID NO:17). In certainembodiments, the biologically-active fragment of factor H fused to ananti-annexin A2 antibody or antigen-binding fragment thereof comprisesSCRs 1 to 4, SCRs 1 to 8, or SCRs 1 to 18 of full-length factor H. Incertain embodiments, the biologically active fragment of MCP fused to ananti-annexin A2 antibody or antigen-binding fragment thereof comprisesthe extracellular domain of full-length human MCP (amino acids 35-343 ofSEQ ID NO:19). In certain embodiments, the biologically active fragmentof MCP fused to an anti-annexin A2 antibody or antigen-binding fragmentthereof comprises SCRs 1 to 4 of full-length human MCP (amino acids35-285 of SEQ ID NO:19). In certain embodiments, the biologically activefragment of CD59 fused to an anti-annexin A2 antibody or antigen-bindingfragment thereof comprises the extracellular domain of full-length humanCD59 (amino acids 26-102 of SEQ ID NO:21) lacking its GPI anchor and/orthe amino acid to which it is attached (i.e., Asn-102).

In certain embodiments, the biologically active fragment of CR1 fused toan anti-annexin A2 antibody or antigen-binding fragment thereofcomprises the complete extracellular domain of full-length human CR1(SCRs 1 to 30)(amino acids 42-1971 of SEQ ID NO:23). In certainembodiments, the biologically active fragment of CR1 fused to ananti-annexin A2 antibody or antigen-binding fragment thereof comprisesSCRs 1 to 4 of full-length human CR1 (amino acids 42-295 of SEQ IDNO:23). In certain embodiments, the biologically active fragment of CR1fused to an anti-annexin A2 antibody or antigen-binding fragment thereofcomprises SCRs 1 to 11 of full-length human CR1 (amino acids 42-745 ofSEQ ID NO:23). In certain embodiments, the biologically active fragmentof CR1 fused to an anti-annexin A2 antibody or antigen-binding fragmentthereof comprises SCRs 1 to 18 of full-length human CR1 (amino acids42-1195 of SEQ ID NO:23). In certain embodiments, the biologicallyactive fragment of mouse Crry protein fused to an anti-annexin A2antibody or antigen-binding fragment thereof comprises the completeextracellular domain of full-length mouse Crry protein (amino acids41-405 of SEQ ID NO:25). In certain embodiments, the biologically activefragment of mouse Crry protein fused to an anti-annexin A2 antibody orantigen-binding fragment thereof comprises short consensus repeatsequences 1 to 5 (SCRs 1 to 5) of full-length mouse Crry protein (aminoacids 83-400 of SEQ ID NO:25). In any of the embodiments disclosedherein, the composition is administered to an individual orally or byinjection. In certain embodiments, the composition is administered to anindividual by intravenous injection. In certain embodiments, thecomposition is administered to an individual by injection into thearteries (such as the renal arteries).

In certain embodiments, alternative complement activity is inhibited inan individual. In certain embodiments, the alternative complementactivity that is inhibited is associated with renal inflammation or adrusen-related disease. In certain embodiments, the renal inflammationis associated with membranoproliferative glomerulonephritis type II(“MPGN II”), hemolytic uremic syndrome (“HUS”), atypical hemolyticuremic syndrome (“aHUS”), thrombotic thrombocytopenic purpura (“TTP”),ischemia/reperfusion injury, or ischemic acute kidney injury. In certainembodiments, the drusen-related disease is selected from the groupconsisting of MPGN II, age-related macular degeneration, andamyloidosis.

In certain embodiments, alternative complement activity is inhibited inan individual. In certain embodiments, the alternative complementactivity that is inhibited is associated with (1) tissue damage due toischemia-reperfusion following acute myocardial infarction, aneurysm,stroke, hemorrhagic shock, crush injury, multiple organ failure,hypovolemic shock intestinal ischemia, spinal cord injury, and traumaticbrain injury; (2) inflammatory disorders, e.g., burns, endotoxemia andseptic shock, adult respiratory distress syndrome, cardiopulmonarybypass, hemodialysis; anaphylactic shock, severe asthma, angioedema,Crohn's disease, sickle cell anemia, poststreptococcalglomerulonephritis, membraneous nephritis, and pancreatitis; (3)transplant rejection, e.g., hyperacute xenograft rejection; (4)pregnancy related diseases such as recurrent fetal loss andpre-eclampsia, and (5) adverse drug reactions, e.g., drug allergy, IL-2induced vascular leakage syndrome and radiographic contrast mediaallergy. In certain embodiments, the alternative complement activitythat is inhibited is associated with autoimmune disorders including, butnot limited to, myasthenia gravis, Alzheimer's disease, multiplesclerosis, rheumatoid arthritis, systemic lupus erythematosus,insulin-dependent diabetes mellitus, acute disseminatedencephalomyelitis, Addison's disease, antiphospholipid antibodysyndrome, autoimmune hepatitis, Crohn's disease, Goodpasture's syndrome,Graves' disease, Guillain-Barré syndrome, Hashimoto's disease,idiopathic thrombocytopenic purpura, pemphigus, Sjögren's syndrome, andTakayasu's arteritis.

In certain embodiments, the alternative complement activity that isinhibited is associated with any of the following disorders: postcardiopulmonary bypass complications; myocardial infarction;ischemia/reperfusion injury; stroke; acute respiratory distress syndrome(ARDS); sepsis; burn injury; inflammation associated withcardiopulmonary bypass and hemodialysis; plasmapheresis;plateletpheresis; leukopheresis; extracorporeal membrane oxygenation(ECMO); heparin-induced extracorporeal LDL precipitation (HELP);radiographic contrast media-induced allergic response; transplantrejection; and other inflammatory conditions and autoimmune/immunecomplex diseases such as multiple sclerosis, myasthemia gravis,pancreatitis, rheumatoid arthritis, Alzheimer's disease, asthma, thermalinjury, anaphylactic shock, bowel inflammation, urticaria, angioedema,vasculitis, glomerulonephritis, and Sjögren's syndrome, systemic lupuserythromatosus and lupus nephritis.

In certain embodiments, alternative complement activity is stimulated inan individual. In certain embodiments, the individual is administered acomposition selected from the group consisting of a biologically-activefragment of factor H lacking the complement regulatory domain in SCRs 1to 4 of full-length factor H. In certain embodiments, the biologicallyactive fragment of factor H comprises SCRs 19 and 20. In certainembodiments, the methods comprise stimulating alternative complement byadministering a composition comprising a biologically-active fragment offactor H comprising SCRs 19 and 20 in conjunction with a monoclonalantibody-based therapeutic. In certain embodiments, the monoclonalantibody-based therapeutic is an anti-cancer agent. In certainembodiments, the alternative complement activity is stimulated in anindividual by administering an effective amount of a compositioncomprising an annexin A2 or a biologically-active fragment thereof. Incertain embodiments, the composition is administered orally or byinjection. In certain embodiments, the composition is injectedintravenously.

The pharmaceutical compositions described herein can be administered toan individual via any route, including, but not limited to, intravenous(e.g., by infusion pumps), intraperitoneal, intraocular, intraarterial,intravesicular, intramuscular, subcutaneous, intrathecal, transpleural,intraarterial, oral, subcutaneous, intraarticular, intracisternal,intraventricular, intracranial, intraurethral, intrahepatic, andintratumoral. In certain embodiments, the pharmaceutical compositionsprovided herein are administered systemically (for example, byintravenous injection). In certain embodiments, the pharmaceuticalcompositions provided herein are administered locally (for example, byintraarterial or intraocular injection).

In certain embodiments, the pharmaceutical compositions provided hereinare administered directly to the eye or the eye tissue. In certainembodiments, the pharmaceutical compositions are administered byinjection to the eye (intraocular injection) or to the tissuesassociated with the eye. The pharmaceutical compositions provided hereincan be administered, for example, by intraocular injection, periocularinjection, subretinal injection, intravitreal injection, transseptalinjection, subscleral injection, intrachoroidal injection, intracameralinjection, subconjunctival injection, subtenon injection, retrobulbarinjection, or peribulbar injection. These methods are known in the art.For example, exemplary periocular routes for retinal drug delivery aredisclosed in “Periocular routes for retinal drug delivery,” Raghava etal., Exp. Opin. Drug Deliv. (2004) 1(1):99-114. The compositionsdescribed herein may be administered, for example, to the vitreoushumor, aqueous humor, sclera, conjunctiva, the area between the scleraand conjunctiva, the retina, the choroid, the macula, to any other areain or proximate to the eye of an individual.

In certain embodiments, the compositions are administeredintravascularly, such as intravenously (IV) or intraarterially. Incertain embodiments (for example for the treatment of renal diseases),the compositions are administered directly into arteries (such as therenal arteries).

EXAMPLES Example 1. Factor H Purification, Analysis and Activities

Materials and Methods

Purification of Mouse Factor H.

An affinity column for factor H was made by ligating polyclonal goatantibody for human factor H (Quidel Corp., San Diego, Calif.) toSepharose® derivatized with cyanogen bromide (CNBr) (AmershamBiosciences/GE Healthcare, Piscataway, N.J.) according to themanufacturer's instructions. Plasma was collected from C57BL/6 mice andpassed over the column. After washing the column with phosphate bufferedsaline (PBS), pH 7.4, the factor H was eluted with 5 M LiCl₂. The saltsolution was exchanged with PBS and the factor H was then passed througha Sephadex™ 26/60 Superdex™ 200 column (Amersham Biosciences/GEHealthcare, Piscataway, N.J.). The purity and identity of the isolatedprotein was verified by Coomassie staining and Western blot analysis bystandard methods.

Western Blot and Far Western Blot Analysis.

Renal tissue was homogenized in radioimmunoprecipitation assay (RIPA)lysis buffer containing 1% Triton® X-100, 0.5% deoxycholic acid, 150 mMNaCl, 20 mM (3-glycerophosphate, 20 mM Tris.HCl (pH 8.0), 5 mM EGTA, 3mM MgCl₂, 0.1% SDS, 1 mM DTT, 50 μM Na₃VO₄, and one tablet of EDTA-freeComplete™ Protease Inhibitor Cocktail (Roche Applied Science,Indianapolis, Ind.). Homogenates were centrifuged at 14,000 rpm for 15minutes at 4° C. and the supernatant collected. Protein concentrationsfor kidney samples were determined using the BioRad protein assay(BioRad Laboratories, Hercules, Calif.). One hundred micrograms ofprotein from each kidney was resolved by electrophoresis on a 10%Bis-Tris polyacrylamide gel (Invitrogen, Carlsbad, Calif.) andtransferred to a nitrocellulose membrane.

Monolayers of proximal tubule epithelial cells (PTECs) were washed withphosphate-buffered saline (PBS) and lysed using 250 l of RIPA bufferapplied to confluent cells on a 24 mm Transwell filter (Corning CostarCorporation, Lowell, Mass.). The lysates were centrifuged at 14,000 rpmfor 15 minutes at 4° C. and the supernatant was collected. Thirtymicroliters of each sample were resolved by electrophoresis on a 10%Bis-Tris polyacrylamide gel (Invitrogen, Carlsbad, Calif.) andtransferred to a nitrocellulose membrane. Factor H was detected in thelysates using a polyclonal goat anti-human antibody (Quidel Corp., SanDiego, Calif.) diluted 1/100 or a monoclonal antibody to mouse factor H(Santa Cruz Biotechnology, Inc., Santa Cruz, Calif.) diluted 1/500.Appropriate secondary antibodies from Jackson ImmunoresearchLaboratories (West Grove, Pa.) were used. Fixation of complement proteinC3 fragments to the surface of cells in culture was detected usingperoxidase-conjugated goat anti-mouse C3 (MP Biomedicals, Santa Ana,Calif.). Antibody binding was detected by exposing the blot tochemiluminescence reagent (Perkin Elmer LAS, Inc., Boston, Mass.). Todetect the binding of factor H to proteins in kidney or cell lysates(Far Western analysis), purified factor H was biotinylated withSulfo-NHS-Biotin (Pierce, Rockford, Ill.) according to themanufacturer's instructions. The sample proteins were separated bySDS-PAGE, transferred to a nitrocellulose membrane, and they wereincubated with 2 μg of biotinylated factor H in PBS, pH 7.4. The bindingof factor H to protein bands was then detected with horseradishperoxidase-conjugated streptavidin (Zymed Laboratories, Inc., SanFrancisco, Calif.) diluted 1/500 in PBS, followed by treatment with theappropriate enzyme chemiluminescence (ECL) substrate.

Protocol for Induction of Renal I/R.

Male C57BL/6J mice (Jackson Laboratories, Bar Harbor, Me.) were used inall experiments. Eight to ten week old mice weighing 20-25 g wereanesthetized with 60 mg/kg ketamine plus 10 mg/kg xylazine (both fromVedco, Inc., St. Joseph, Mo.) injected intra-peritoneally. Mice wereplaced on a heating pad to maintain their body temperature duringsurgery. Laparotomies were then performed and the renal pedicles werelocated and isolated by blunt dissection as previously described. See.M. Thurman et al., J. Immunol. (2003) 170:1517-1523. The pedicles wereclamped with surgical clips (Miltex Instrument Company, Inc., York,Pa.), and occlusion of blood flow was confirmed by visual inspection ofthe kidneys. The clamps were left in place for 24 minutes and thenreleased. The kidneys were observed for approximately one minute toensure blood re-flow, then fascia and skin were sutured with 4-0 silk(United States Surgical Corp., Princeton, N.J.). Sham surgery wasperformed in an identical fashion, except that the renal pedicles werenot clamped. The mice were volume resuscitated with 0.5 ml of normalsaline and kept in an incubator at 29° C. to maintain body temperature.After 8 or 24 hours the mice were anesthetized, and blood was obtainedby cardiac puncture. Laparotomy was performed and the kidneys wereharvested. All animal procedures were in adherence to the NationalInstitutes of Health Guide for the Care and Use of Laboratory Animals.

In order to suppress renal production of annexin 2 after I/R, C57BL/6mice were injected with antisense oligodeoxynucleotides (ODNs) againstannexin 2. The mice were injected with a mixture containing 5 nmol of 3different antisense ODNs (Biognostik Reference Nos. 1.06412, 2.06413,3.06414) as known in the art and described in Table 1 below, or controlODNs (Biognostik's “Random Control I” and “Random Control II”). TheseODNs were designed and manufactured by Biognostik (Gottingen, Germany).To confirm tubular uptake of ODNs, mice received intraperitonealinjections of 5 nmol fluorescein isothiocyanate-labeled (FITC-labeled)ODN (Biognostik, Gottingen, Germany).

TABLE 1 Antisense Oligonucleotides to Annexin A2 Sequence SEQ ReferenceSequence ID NO: 1.06412 5′-TCT CCA GCA TGT CAT AAG-3′ 27(784-801 bases of total sequence) 2.06413 5′-GTC TGC CCT TTG CAA G-3′ 28(594-609 bases of total sequence) 3.06414 5′-CAA TGT CCT GCC TCT G-3′ 29(273-288 bases of total sequence)

Immunofluorescence, Immunohistochemistry and Direct Binding of Factor Hto Tissue Sections.

Sagittal sections of the kidneys were snap frozen in Tissue-Tek^(R)optimal cutting temperature (OCT) compound (Sakura Finetek, La Jolla,Calif.). Four micrometer sections were cut with a cryostat and stored at−70° C. The slides were later fixed with acetone. Complement protein C3was detected with a FITC-conjugated anti-mouse C3 antibody (MPBiomedicals, Santa Ana, Calif.) diluted 1:150. Factor H was detectedwith a polyclonal goat anti-human antibody (Quidel Corp., San Diego,Calif.) diluted 1:200 or a monoclonal antibody to mouse factor H (SantaCruz Biotechnology, Inc., Santa Cruz, Calif.) diluted 1:200. To detectannexin A2 expression, sections were incubated with a monoclonalantibody for mouse annexin A2 (Zymed Laboratories, Inc., San Francisco,Calif.). Appropriate secondary antibodies from Jackson ImmunoResearchwere used. Biotinylated mouse factor H was directly bound to tissuesections by diluting the protein in PBS and incubating it on sectionsfor one hour. The sections were then incubated with FITC-conjugatedstreptavidin (Zymed Laboratories, Inc., San Francisco, Calif.). In allof these procedures tissue sections were counterstained with hematoxylin(Vector Laboratories, Burlingame, Calif.).

Direct Binding of Factor H to Cells and to Immobilized Annexin A2.

To determine whether factor H binds directly to cells, mouse PTECs weregrown on Transwell filters until stable trans-epithelial resistance wasobtained. Biotinylated factor H was added to either the apical or basalchamber and incubated for one hour. The cells were then washed with PBSand protein lysates were made as described above. The proteins wereseparated by SDS-PAGE, and bound factor H was detected usingHRP-conjugated streptavidin (Zymed Laboratories, Inc., San Francisco,Calif.), followed by chemi-luminescent detection. To determine whetherpurified factor H bound directly to annexin A2, enzyme-linkedimmunosorbent assay (ELISA) plates were coated with 100 ng of purifiedbovine annexin A2 (Biodesign, Saco, Me.) diluted in 15 mM Na₂CO₃/35 mMNaHCO₃. After blocking the plates with 1% BSA for 1 hour, 500 ng ofbiotinylated factor H diluted in PBS containing 3 mM CaCl₂ was added tothe wells. Unbound factor H was washed off using the same buffer, andbound factor H was detected using HRP-conjugated streptavidin (Zymed,San Francisco, Calif.) followed by 50 μl ABTS solution(ABTS=2,2′-azino-di-(3-ethylbenzthiaoline sulfonic acid). Absorbance wasread at 405 nm.

Mass Spectroscopy.

Binding partners for factor H were isolated by pull-down experiments asdescribed above. After separating the pulled-down proteins by SDS-PAGEand staining them with Coomassie Blue, a band of approximately 39kilodaltons (kD) molecular weight was identified. Bands were excised andproteins digested in the gel using a modification of the methods ofHavlis (J. Havlis et al., “Fast-response proteomics by acceleratedin-gel digestion of proteins,” Anal. Chem. (2003) 75:1300-1306) andJimenez (C. Jimenez et al., IN-GEL DIGESTION OF PROTEINS FOR MALDI-MSFINGERPRINT MAPPING (John Wiley & Sons, Inc.: Brooklyn, N.Y. (1998)), atpp. 16.4.1-16.4.5). Briefly, bands were crushed using a closed pipettetip and destained twice with a 1:1 mixture of acetonitrile and 100 mMammonium bicarbonate, then contacted with 100% acetonitrile and driedunder vacuum. Samples were reduced with freshly prepared dithiothreitol(1.5 mg/ml) and incubated at 37° C. for 60 minutes. After cooling to 4°C., samples were alkylated in the dark for 45 minutes using a 10 mg/mlsolution of iodoacetamide. After washing in a 1:1 mixture ofacetonitrile and 100 mM ammonium bicarbonate, samples were dried undervacuum, transferred to clean tubes, and rehydrated in trypsin at 0.2mg/ml in 50 mM ammonium bicarbonate at 37° C. overnight. Thesupernatants were then sonicated at room temperature after the additionof 1-2 μl of formic acid, collected, and pooled with one additionalextract prepared using 0.1% aqueous trifluoroacetic acid with 60%acetonitrile. Pooled extracts were vacuum-concentrated to a volume ofapproximately 10 l and stored at −80° C. until used.

LC-MS/MS analysis of enzymatic digests.

Tandem mass spectroscopy analysis was performed at the Mass SpectrometryCore Facility at the University of Colorado Health Sciences Center.Approximately 30% of each in-gel digested samples were analyzed byreverse phase nanospray LC-MS/MS using an Agilent 1100 HPLC 75 m×15 cmcolumn packed with 5 m Zorbax C18 particles). Spectra were collectedover a m/z range from 350 to 1800 daltons (Da) (Agilent LC/MSD Trap XCTUltra). Active exclusion was used to access lower abundance peptides,where six precursor ions were selected for fragmentation and thenexcluded from analysis after two measurements.

Surface Plasmon Resonance.

Factor H was immobilized on a CM5 sensor chip. Bovine annexin A2 wasdiluted in Hank's Buffered Saline with or without 3 mM CaCl₂ andinjected at the following concentrations: HBS alone (no protein)+3 mMCaCl₂ (second line from bottom-no binding); 3.7 μg/ml in HBS+3 mM CaCl₂(third line from bottom); 7.5 μg/ml in HBS+3 mM CaCl₂ (fourth line frombottom); 15 μg/ml in HBS+3 mM CaCl₂ (fifth line from bottom); 30 μg/mlin HBS+3 mM CaCl₂ (top line); and 30 μg/ml in Hank's Buffered Salinewithout calcium (bottom essentially constant line—no binding). Theanalyte bound to the chip with high affinity (17 nm), but rapidlydissociated from the chip when calcium was removed from cell.

Statistics.

Multiple group comparisons were performed using analysis of variance(ANOVA) and post-test according to Newman-Keuls, using the GraphPadPrism™ software package (GraphPad Software, Inc., La Jolla, Calif.). A Pvalue of less than 0.05 was considered statistically significant.Results are reported as mean±standard error (SE).

Results

Factor H Limits Complement Mediated Injury after Renal I/R.

Complement protein C3 is deposited along the basolateral aspect oftubules in the outer medulla after renal I/R. J. M. Thurman et al.,(2006) J. Clin. Invest. 116:357-368; W. Zhou et al., (2000) J. Clin.Invest. 105:1363-1371. To test whether circulating factor H isfunctionally important after renal I/R, we subjected mice to renal I/Rthen injected them with a recombinant protein that competitively blocksbinding of factor H at the carboxy terminus. Mice injected with thisprotein showed greater systemic C3a than controls (FIG. 1A) after 8hours of reperfusion. Levels were no different than control animalsinjected with an equal volume of PBS by 24 hours of reperfusion (FIG.1B), suggesting that factor H is functionally blocked within 8 hours ofreperfusion in this model. Renal I/R typically causes injury of thetubules in the outer medulla (FIG. 1C). In mice treated with rH19-20 theinjured tubules extended into the cortex (FIG. 1D). Serum urea nitrogen(SUN) levels were also significantly higher in mice treated with rH19-20(FIG. 1E), indicating worse functional impairment in mice treated withrH19-20. Although circulating factor H does not fully prevent complementactivation in the kidney after I/R, this experiment demonstrates that itdoes function to limit complement-mediated injury during reperfusion.

Native Factor H does not Protect PTECs at Baseline.

PTECs were grown in primary culture. The cells were then incubated with10% normal mouse serum in the presence or absence of rH19-20. Finally,the cells were stained with an FITC-conjugated antibody to mouse C3protein, and complement activation on the cell surface was measured byFACS analysis. Treatment with rH19-20 did not cause increased depositionof complement protein C3.

Factor H Accumulates in the Tubulointerstitium after I/R but does notCo-Localize with C3.

Given the functional importance of factor H during reperfusion, we nextexamined the kidneys for the presence of factor H. C3 deposits are seenon injured tubules within 6 hours of I/R and peak after 24 hours ofreperfusion in our model. See J. M. Thurman et al., J. Clin. Invest.(2006) 116:357-368. Western blot analysis of kidney lysates demonstratesthat factor H levels within the kidney rise within 8 hours ofreperfusion (FIG. 2A). It has been proposed that factor H binds to C3bdeposited on anionic surfaces (S. Meri et al., Proc. Nat'l Acad. Sci.USA (1990) 87:3982-3986; S. Meri et al., Biochem. Biophys. Res. Comm.(1994) 198:52-59, but factor B deficient mice subjected to renal I/R [inwhich no C3 is deposited on the tubules after I/R (J. M. Thurman et al.,J. Immunol. (2003) 170:1517-1523)] demonstrated factor H levelsequivalent to those in wild-type mice after renal I/R (FIG. 2B).Furthermore, dual staining for factor H and C3d (FIGS. 3A-3F)demonstrated that during reperfusion the tubulointerstitial factor Hdoes not co-localize with C3d. This may be due to the ability of thebound factor H to prevent AP activation, but it suggests that factor Hdoes not require C3d in order to bind within post-ischemic kidney whenother appropriate ligands are present.

Annexin 2 is Expressed in the Kidneys During Reperfusion and is aBinding Ligand for Factor H.

To determine whether protein ligands may mediate the increased bindingof factor H seen during reperfusion, we performed Far-Western analysisof kidney lysates using purified factor H (FIG. 4A), and found thatfactor H bound to proteins present in the post-ischemic lysates thatwere not evident in the lysates of unmanipulated kidneys. We used factorH to pull down binding partners in the lysates (FIG. 4B), and againfound that new binding partners were present in the lysates ofpost-ischemic kidneys.

We examined the ˜39 kD binding partner by tandem mass spectroscopy (FIG.4C) and identified the protein as annexin A2. Typically, at least 2peptide hits are sought to confirm a protein's identity. We obtained 6unique peptides for annexin A2, and three were manually confirmed. FIG.4C shows the MS spectrum for one of the peptides. The mass of thepeptide is 1422.0527 a.m.u., and the observed peak chosen forfragmentation was at m/z 711.53, corresponding to a doubly-charged ion.The peptide sequence is shown and the b- and y-type fragment ions thatwere observed are indicated by a line over or under the letterrepresenting the amino acid. The data was searched against the SwissProtdatabase (human mouse taxonomy) using SpectrumMill from AgilentTechnologies (Santa Clara, Calif.). The search score was 18.91. The datawas also searched against a reverse database and that score was 0,indicating that the data is of very good quality. The single photonionization percentage (SPI %) (a measure of how much of the ion currentis accounted for by theoretically expected peaks) for this spectrum was92.6, suggesting that the dominant ions conform to those expected forthis sequence.

Annexin A2 is a Ca²⁺-regulated phospholipid binding protein. See V.Gerke et al., Physiol. Rev. (2002) 82:331-371. Extracellular annexin A2functions as a surface bound receptor for several different molecules,including plasminogen and tissue plasminogen activator. See K. A. Hajjaret al., J. Biol. Chem. (1994) 269:21191-21197. It has previously beenreported that annexin A2 is expressed by regenerating tubules aftertoxic and ischemic injury of the kidney. See C. W. Cheng et al., KidneyInt'l (2005) 68:2694-2703. We detected annexin A2 in the glomeruli andin injured renal tubules after I/R (FIG. 5A). Western blot analysis ofproteins pulled down by purified factor H using a monoclonal antibodyfor annexin A2 confirmed that factor H bound to annexin A2 in thelysates of post-ischemic kidneys (FIG. 5B). When a recombinant fragmentof factor H containing short consensus repeats 19 and 20 (rH 19-20) wasadded to the pull-down reaction it blocked the binding of factor H andannexin A2 (FIG. 5B), indicating that this protein-protein interactioninvolves the carboxyl-terminus of factor H.

Factor H Binds Directly to Annexin A2 in a Calcium Dependent Fashion.

To determine whether factor H binds directly to annexin A2 or whetherbridging molecules are required, surface plasmon resonance wasperformed. Factor H was coupled to a CM5 chip, and bovine annexin A2 wasintroduced. The annexin A2 bound to the chip with high affinity in thepresence of calcium-containing buffer. When the buffer was changed to acalcium-free buffer, the annexin A2 rapidly dissociated from the chip.Control protein (annexin A5) did not bind to the factor H-coupled chip.

Targeted Factor H Prevents Complement Activation after Renal I/R.

Based upon the above findings, we hypothesized that the inability ofnative factor H to prevent complement activation within the kidney wasdue to insufficient expression of binding ligands at this location. Toovercome this limitation we employed a recombinant protein that targetsthe inhibitory region of mouse factor H specifically to sites of C3ddeposition. This protein is comprised of the C3d binding region ofcomplement receptor 2 linked to the first four SCRs (the complementinhibitory region) of factor H (referred to as “CR2-fH”), a strategythat has previously been utilized for the targeting of complementinhibitors to sites of complement activation. See H. Song et al., J.Clin. Invest. (2003) 111:1875-1885; C. Atkinson et al., J. Clin. Invest.(2005) 115:2444-2453. Since they contain the same inhibitory region, anyfunctional superiority of the CR2-fH compared with endogenous factor His most likely attributable to differential binding of the protein toC3d on the specific surface in question.

Several in vitro assays are used to assess alternative pathwayactivation. The lysis of erythrocytes by normal human serum iscritically dependent on the interaction of factor H in the serum withanions on the erythrocyte surface. See V. P. Ferreira et al., J.Immunol. (2006) 177:6308-6316. Sheep erythrocytes, for example, are moresensitive to lysis by human serum than human erythrocytes are because ofa stronger interaction of factor H with the human cells. Zymosanparticles have frequently been used to activate the alternative pathwayin vitro, and abundant C3 is deposited on the particles when incubatedwith serum. See J. M. Thurman et al., Mol. Immunol. (2005) 42:87-97.Although factor H present in the serum does not prevent activation ofthe alternative pathway on the zymosan surface (M. K. Pangburn et al.,J. Immunol. (2000) 164:4742-4751), the CR2-targeted factor H does (FIGS.4A-4B).

We subjected mice to renal I/R and injected them with 150 μg ofCR2-targeted factor H after 15 minutes of reperfusion. We found that theCR2-targeted factor H attenuated tubulointerstitial complementactivation after renal I/R (FIGS. 7A-7C) and ameliorated renal injury(FIG. 7D). Thus, the targeted factor H more effectively preventedcomplement activation on the post-ischemic kidney than native factor H.

Targeted Factor H Prevents Fixation of C3 to the Basolateral Surface ofHypoxic PTECs.

PTECs were next grown on Transwell™ filters until they formed a stablemonolayer. Chemical hypoxia was induced by treating the cells withAntimycin A. See J. M. Thurman et al., J. Am. Soc. Nephrol. (2006)17:707-715. The cells were then exposed to 10% serum for one hour, andlysates of the cells were prepared. Western blot analysis for complementprotein C3 demonstrated that C3 degradation fragments were fixed to thecell surface in spite of the factor H in the serum. The addition ofCR2-fH to the serum prevented fixation of C3 to the cells, but theaddition of superphysiologic concentrations of native factor H did not.

Discussion

Circulating factor H does not prevent alternative pathway activation inthe kidney after I/R. We found, however, that levels of factor H in thekidney increased during reperfusion, and that treatment with acompetitive inhibitor of the binding region of factor H caused greatercomplement activation and greater renal injury after I/R. These resultsindicate that native factor H does effectively limit alternative pathwayactivation during reperfusion. Although factor H contains binding sitesfor C3d, the tissue bound factor H did not co-localize with C3d, andfactor H was also accumulated in the kidneys of complement deficientmice subjected to renal I/R. Furthermore, proteomic analysis indicatedthat other proteins expressed in the post-ischemic kidney, such asannexin A2, can bind factor H with high affinity. Thus, factor H doesnot prevent complement activation in the kidney after I/R, but thegeneration of factor H binding ligands in post-ischemic tissue is acritical mechanism by which inflammation is limited.

We next treated mice with a recombinant protein that specificallytargets factor H to sites of deposited C3d (CR2-fH). This agent almostcompletely prevented tubulointerstitial complement activation afterrenal I/R and ameliorated the development of renal injury. Thus, thisagent was much more effective than endogenous factor H at preventingalternative pathway activation after renal I/R. This finding furtherillustrates that complement inhibition by native factor H in the kidneyis limited by the protein's affinity for this surface. We specificallyexamined the interactions between factor H and hypoxic tubularepithelial cells because complement activation on this surface occurspredominantly through the alternative pathway. Our results, however,have several important implications regarding the general mechanisms bywhich the complement system is controlled.

The ability of factor H to discriminate between host cells and invasivepathogens has been attributed to binding of the factor H molecule tonegatively charged molecules such as sialic acid and glycosaminoglycansthat are displayed on the surface of host cells. See S. Meri et al.,Proc. Nat'l Acad. Sci. USA (1990) 87:3982-3986. In diseases such as AMDand aHUS the anatomic restriction of disease has been attributed to: i)efficient complement inhibition by factor H on the negatively chargedbasement membranes in the eye and kidney, and ii) the absence of othercomplement regulatory proteins on these surfaces. The factor H variantsthat are strongly associated with the risk of these diseases demonstratereduced affinity for polyanions (G. S. Hageman et al., Proc. Nat'l Acad.Sci. USA (2005) 102:7227-7232; A. P. Sjoberg et al., J. Biol. Chem.(2007) 282:10894-10900; M. C. Pickering et al., Clin. Exp. Immunol.(2008) 151:210-230), perhaps explaining the predisposition to disease inthe eye and kidney. The onset of AMD is usually in the 6^(th) decade oflife, however, and aHUS can be triggered by infections or other types ofendothelial injury (M. C. Pickering and H. T. Cook, “Translationalmini-review series on complement factor H: renal diseases associatedwith complement factor H—novel insights from humans and animals,” Clin.Exp. Immunol. (2008) 151:210-230). Furthermore, aHUS is also associatedwith congenital mutations in MCP. These observations suggest complexinteractions between factor H and host tissues.

Little, however, is known about different binding ligands for factor Hexpressed within these tissues. Our data indicates that surface proteinsalso mediate the binding of factor H to host cells, and that the cellsactively modulate expression of these binding ligands. In addition topolyanions, the affinity of factor H for each tissue type may bedetermined by its interactions with numerous different binding ligands.

The alternative pathway of complement has emerged as an importanttrigger of inflammation in a number of different diseases. See J. M.Thurman et al., J. Immunol. (2006) 176:1305-1310. Pathologic alternativepathway activation is frequently due to congenital or acquired defectsin the proteins that regulate the complement system. Cellular injury mayalso foster alternative pathway activation by reducing local expressionof complement regulatory proteins, suggesting that complement inhibitionon a given tissue or cell type is not a static characteristic of thatsurface, but may significantly change during injury or recovery.

Distinct tissues express different combinations of the membrane boundcomplement inhibitors, and factor H likely has different affinities forvarious cell types. Consequently, congenital defects in complementregulatory proteins may render particular organs susceptible tocomplement mediated injury, such as the eye (G. S. Hageman et al., Proc.Nat'l Acad. Sci. USA (2005) 102:7227-7232; A. P. Sjoberg et al., J.Biol. Chem. (2007) 282:10894-10900) and the kidney (M. C. Pickering etal., J. Exp. Med. (2007) 204:1249-1256; M. C. Pickering et al., NatureGenet. (2002) 31(4):424-428). Based upon the results presented in thecurrent study, a reduction or increase in the affinity expression ofligands for factor H may also underlie the activation or control of thealternative pathway in disease.

Example 2. Effect of Annexin 2 During Renal Failure

Experimental

Renal Ischemia/Reperfusion Protocol.

Eight to ten week old male C57BL/6J mice (Jackson Laboratories) miceweighing 20-25 g were anesthetized with 60 mg/kg ketamine plus 10 mg/kgxylazine (Vedco, Inc., St. Joseph, Mo.) injected intra-peritoneally.Mice were placed on a heating pad to maintain body temperature duringsurgery. Laparotomies were then performed and the renal pedicles werelocated and isolated by blunt dissection as known in the art. Thepedicles were clamped with surgical clips (Miltex Instrument Company),and occlusion of blood flow was confirmed by visual inspection of thekidneys. The clamps were left in place for 24 minutes and then released.The kidneys were observed for approximately one minute to ensure bloodre-flow, then fascia and skin were sutured with 4-0 silk (United StatesSurgical). Sham surgery was performed in an identical fashion, exceptthat the renal pedicles were not clamped. The mice were volumeresuscitated with 0.5 ml of normal saline and kept in an incubator at29° C. to maintain body temperature. After eight or 24 hours the micewere anesthetized, and blood was obtained by cardiac puncture.Laparotomy was performed and the kidneys were harvested.

Knock-Down of Annexin A2 Production In Vivo Using Anti-SenseOligonucleotides.

In order to suppress renal production of Annexin 2 after I/R, C57BL/6mice were injected with antisense oligodeoxynucleotides (ASOs) againstAnnexin 2. The mice were injected intra-peritoneally with a mixturecontaining 5 nmol of 3 different antisense ASOs or control ODNs. SeeTable 1 herein. These ODNs were designed and manufactured by Biognostik(Göttingen, Germany). Mice received injections of the ASOs two daysprior to undergoing renal I/R and on the morning of renal I/R.

Measurement of C3a.

Plasma C3a was measured as a marker of complement activation in micesubjected to renal I/R after treatment with ASO to Annexin A2 or withcontrol ASO. Plasma samples were taken from mice after 8 hours ofreperfusion, and C3a levels were measured by enzyme linked immunosorbentassay (ELISA) according to the manufacturer's instructions (BDPharmingen). Briefly, ELISA platers were coated with 200 ng of captureantibody as known in the art. After blocking the plates with 1% bovineserum albumin, the plasma samples were diluted 1/50 in PBS and appliedto the plates for 1 hour at room temperature. A detection antibody wasthen applied to the plate (100 l of antibody diluted to 1.0 μg/mL),followed by detection with streptavidine-HRP (diluted 1:2500) and ABTS.Fluorescence was then read at 405 nm on a plate reader.

In Vitro Analysis of Complement Activation on Tubular Epithelial Cells.

To measure complement activation on the surface of renal tubularepithelial cells (TECs), murine TECs were grown to confluence. The cellswere released from the plates by treatment with Accutase® (InnovativeCell Technologies, Inc.) and washed in PBS. For complement activationexperiments, the cells were then incubated in 10% mouse serum at 37° C.for 20 minutes. In some experiments 10 μg of rH 19-20 or 11.2 μg ofAnnexin A2 was added to the reaction. The cells were then washed in PBSand incubated with a FITC conjugated antibody to mouse C3. Cells werethen washed and resuspended in 500 μL of PBS, run on a FACSCalibur™machine (BD Biosciences), and the results were analyzed with CellQuestPro™ software (BD Biosciences). Surface fluorescence for C3 wasinterpreted as complement activation on the cell surface.

Results

As shown in FIG. 8, mice in which Annexin A2 production is blockeddisplay greater complement activation after renal I/R. Mice were treatedwith ASOs to Annexin A2 or with control ASOs, and were then subjected torenal I/R. After eight hours of reperfusion plasma samples wereobtained, and C3a was measured by ELISA. Levels of C3a in mice treatedwith ASOs to Annexin A2 were higher than those in control animals.Without wishing to be bound by any theory, these results demonstratethat Annexin A2 functions to limit complement activation after renalI/R.

As shown in FIG. 9, mice in which Annexin A2 production is blockeddisplay more severe renal injury after renal I/R than control mice. Micewere treated with ASO to Annexin A2 or with control ASOs, and were thensubjected to renal I/R. After twenty-four hours of reperfusion the micewere sacrificed, and serum urea nitrogen (SUN) was measured as a markerof renal function. SUN levels in mice treated with ASOs to Annexin A2were higher than those in control animals. Without wishing to be boundby any theory, the results demonstrate that Annexin A2 functions tolimit renal injury after renal I/R. Furthermore, SUN levels incomplement deficient mice (deficient in factor B, or fB−/− mice) thatwere treated with the ASOs to Annexin A2 were not higher than controlmice, indicating that the ASOs to Annexin A2 require an intactcomplement system in order to cause renal injury after I/R.

As shown in FIG. 10, purified Annexin A2 can enhance complementactivation on cell surfaces by blocking the interaction of factor H withthe cell surface. Renal tubular epithelial cells were grown in cultureand were then exposed to 10% mouse serum (shaded curve). Treatment ofthe cells with rH19-20 blocked protection of the cells by factor Hpresent within the serum, thereby enhancing the deposition of C3 on thecell surface (solid line). The addition of purified Annexin A2 to thecell supernatant had a similar effect (dashed line). The addition ofboth rH19-20 and Annexin A2 caused a similar degree of complementactivation on the cells than was obtained with either reagent alone.Without wishing to be bound by any theory, these data can be interpretedas reflecting a competition of factor H away from the cell surface dueto Annexin A2.

Although the foregoing has been described in some detail by way ofillustration and example for purposes of clarity of understanding, it isapparent to those skilled in the art that certain changes andmodifications will be practiced without departing from the disclosedscope. Therefore, the description and examples should not be construedas limiting the scope provided herein.

What is claimed is:
 1. A method of treating complement-mediatedinflammation, the method comprising administering to a subject apharmaceutical composition comprising a fusion protein that comprises abiologically-active fragment of at least one of: complement receptor 1(CR1), decay-accelerating factor (DAF), factor H, Membrane cofactorprotein (MCP), CD59, and Crry protein, fused to an antibody orantigen-binding fragment thereof that binds annexin A2, wherein saidadministering is in an amount sufficient to treat thecomplement-mediated inflammation.
 2. The method of claim 1, wherein saidsubject is a human.
 3. The method of claim 1, wherein saidcomplement-mediated inflammation is associated with uncontrolledalternative pathway activation.
 4. The method of claim 1, wherein saidcomplement-mediated inflammation is associated with a condition ordisease.
 5. The method of claim 4, wherein said condition or disease isselected from the group consisting of ischemia/reperfusion injury, burninjury, endotoxemia and septic shock, adult respiratory distresssyndrome, cardiopulmonary bypass, hemodialysis, anaphylactic shock,asthma, angioedema, Crohn's disease, sickle cell anemia,glomerulonephritis, membraneous nephritis, pancreatitis, transplantrejection, hyperacute xenograft rejection, recurrent fetal loss,preeclampsia, drug allergy, IL-2 induced vascular leakage syndrome,radiographic contrast media allergy, myasthenia gravis, Alzheimer'sdisease, multiple sclerosis, rheumatoid arthritis, systemic lupuserythematosus, insulin-dependent diabetes mellitus, acute disseminatedencephalomyelitis, Addison's disease, antiphospholipid antibodysyndrome, autoimmune hepatitis, Goodpasture's syndrome, Graves' disease,Guillain-Barre syndrome, Hashimoto's disease, idiopathicthrombocytopenic purpura, pemphigus, Sjogren's syndrome, Takayasu'sarteritis, myocardial infarction, stroke, acute respiratory distresssyndrome, sepsis, plasmapheresis, plateletpheresis, leukopheresis,extracorporeal membrane oxygenation, heparin-induced extracorporeal LDLprecipitation, bowel inflammation, urticarial, and vasculitis and lupusnephritis.
 6. The method of claim 1, wherein the complement-mediatedinflammation is associated with a condition or disease selected from thegroup consisting of age-related macular degeneration (AMD), type IImembranoproliferative glomerulonephritis (MPGN II), hemolytic uremicsyndrome (HUS), asthma, amyloidosis, and thrombotic thrombocytopenicpurpura.
 7. The method of claim 6, wherein said hemolytic uremicsyndrome (HUS) is an atypical hemolytic uremic syndrome (aHUS).
 8. Themethod of claim 1, wherein said pharmaceutical composition isadministered by intravenous injection.
 9. A method of treatingcomplement-mediated inflammation associated with a drusen-associateddisease or a drusen-related disease, the method comprising administeringto a subject a composition comprising a polypeptide construct, in anamount sufficient to treat complement-mediated inflammation associatedwith said drusen-associated disease or drusen-related disease, whereinsaid polypeptide construct comprises a biologically-active fragment ofat least one of: CR1, DAF, factor H, MCP, CD59, and Crry protein fusedto an antibody or antigen-binding fragment thereof that binds annexinA2.
 10. The method of claim 9, wherein said drusen-related disease isamyloidosis, elastosis, dense deposit disease, glomerulonephritis,atherosclerosis or an ocular drusen-related disease.
 11. The method ofclaim 9, wherein said polypeptide construct comprises abiologically-active fragment of at least one of: factor H, CR1, and DAFfused to the antibody or a single chain variant fragment thereof,wherein said antibody or single chain variant fragment thereof bindsannexin A2.
 12. The method of claim 11, wherein said drusen-relateddisease is an ocular drusen-related disease.
 13. The method of claim 11,wherein said drusen-related disease is a non-ocular drusen-relateddisease.
 14. The method of claim 13, wherein said non-oculardrusen-related disease is amyloidosis, elastosis, dense deposit disease,glomerulonephritis or atherosclerosis.
 15. A method of treatingcomplement-mediated inflammation associated with a disease, the methodcomprising administering to a subject a pharmaceutical compositioncomprising a polypeptide construct, in an amount sufficient to treatcomplement-mediated inflammation associated with said disease, whereinsaid disease is age-related macular degeneration (AMD), type IImembranoproliferative glomerulonephritis (MPGN II), hemolytic uremicsyndrome (HUS), asthma, amyloidosis, glomerulonephritis, membraneousnephritis, transplant rejection, antiphospholipid antibody syndrome,pemphigus, stroke, bowel inflammation, lupus nephritis, oculardrusen-related disease, dense deposit disease or thromboticthrombocytopenic purpura, wherein said polypeptide construct comprises abiologically-active fragment of at least one of: factor H, CR1, and DAFfused to an antibody or a single chain variant fragment thereof, whereinsaid antibody or single chain variant fragment thereof binds annexin A2.16. The method of claim 15, wherein said hemolytic uremic syndrome (HUS)is an atypical hemolytic uremic syndrome (aHUS).
 17. The method of claim16, wherein said antibody or single chain variant fragment thereof bindsan annexin core domain.
 18. The method of claim 15, wherein said subjectis human.
 19. The method of claim 15, wherein said pharmaceuticalcomposition is administered orally, or by intravenous injection.
 20. Amethod of treating glomerulonephritis having complement-mediatedinflammation, the method comprising administering to a subject acomposition comprising a polypeptide construct, in an amount sufficientto treat glomerulonephritis having complement-mediated inflammation,wherein said polypeptide construct comprises a biologically-activefragment of at least one of: complement receptor 1 (CR1),decay-accelerating factor (DAF), factor H, Membrane cofactor protein(MCP), CD59, and Crry protein fused to an antibody or antigen-bindingfragment thereof that binds annexin A2.